Single-component RNA vectors derived from a virus and containing an intervening sequence between the cap and the 5&#39; end and able to replicate in a host plant cell within a host plant

ABSTRACT

This invention is directed to a plus strand RNA viral vector for transformation of a host organism with a foreign RNA, and expression of said foreign RNA. The foreign RNA is inserted into an infective RNA viral segment containing cis-acting viral replication elements, and allowed to infect the host organism. The RNA vector is modified to obtain infectivity by including an intervening sequence between the cap and the 5′ end. The modified RNA is able to tolerate the exogenous RNA segment without disrupting the replication of the modified RNA, in the absence of a trans-acting viral replication element in a single component plant virus host cell.

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/949,316, which is a continuation of U.S. Pat.No. 6,300,134 issued Oct. 9, 2001, which is a continuation-in-part ofU.S. patent application Ser. Nos. 09/359,301 and 09/359,305, filed Jul.21, 1999, which are continuations-in-part of U.S. patent applicationSer. No. 09/232,170, filed Jan. 15, 1999, which is acontinuation-in-part of U.S. patent application Ser. No. 09/008,186,filed Jan. 16, 1998. The above parent applications are incorporatedherein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to the field of plant viruses, moreparticularly to plus-sense RNA plant virus, and to modifications, madeaccording to the teachings herein, which permit insertion of anexogenous RNA segment into the viral genome. The recombinant plant viralnucleic acid construct derived from insertion of an exogenous RNAsegment into the viral genome can then be introduced into a host cell inorder to modify the cell.

BACKGROUND OF THE INVENTION

[0003] RNA viruses whose genome is composed of a single RNA strandcapable of replication in the cytoplasm of a host by direct RNAreplication are widespread, many varieties of which are known to infectplants. Such viruses are sometimes termed “(+) strand RNA viruses” sincethe infective RNA strand, that normally found encapsidated in the virusparticle, is a messenger-sense strand, capable of being directlytranslated, and also capable of being replicated under the properconditions by a direct process of RNA replication. Viruses belonging tothis group include “single component (+) strand RNA viruses”, whichreplicate in the absence of trans-acting viral replication elements.These viruses may include, but are not limited to any of therepresentatives of the following virus groups, Carlavirus,Closteroviridae, Luteoviridae, Potexvirus, Potyviridae, Tombusviridae,Tobamovirus and Tymovirus. (Similar viruses, which in the host cellproduce a trans-acting replication element, are not included in thisgroup.) In these cases, the entire virus genome is contained within asingle RNA molecule, while in the multicomponent RNA plant viruses, thetotal genome of the virus consists of two or more distinct RNA segments,each separately encapsidated. For general review, see General Virology,S. Luria and J. Darnell; Plant Virology 2nd ed., R. E. F. Matthews,Academic Press (1981). For a general review of (+) strand RNAreplication, see Davies and Hull (1982) J. Gen. Virol. 61:1.

[0004] Despite the well-documented diversity between virus groups,recent studies have shown striking similarities between the proteins,which function in RNA replication. Sequence homologies have beenreported between the cowpea mosaic virus, poliovirus and foot-and-mouthdisease virus, (Franssen, H. (1984) EMBO Journal 3,855). Sequencehomologies have been reported between non-structural proteins encoded byalfalfa mosaic virus, brome mosaic virus and tobacco mosaic virus,Haseloff, J. et al. (1984), Proc. Nat. Acad. Sci. USA 81, 4358, andbetween non-structural proteins encoded by sindbis virus, Ahlquist, P.et al. (1985) J. Virol. 53, 536. Evidence of such substantial homologyin proteins related to the replication functions indicate that theviruses share mechanistic similarities in their replication strategiesand may actually be evolutionarily related. Ahlquist et al., in U.S.Pat. No. 5,500,360 made modifications to the genomic RNA of a (+) strandRNA virus of a multipartite Brome mosaic virus. The modified RNA wasused to transfer a desired RNA segment into a targeted host plantprotoplast, and to replicate that segment and express its functionwithin the host protoplast.

[0005] In contrast to the Brome mosaic virus (BMV), the tobacco mosaicvirus (TMV) is one member of a class of plant viruses characterized by asingle RNA genome. The genetic material of the virus is RNA, and thetotal genetic information required for replication and productiveinfection is contained in one discrete RNA molecule. Infection of a hostplant cell occurs when the single RNA component of the viral genome hasinfected the cell, for example by exposing a plant to a viruspreparation. Infection may also be achieved by exposing a plant cell orprotoplast to a virus preparation. TMV does not require coat protein forinfection. The RNA component is both necessary and sufficient forreplication and productive infection. The TMV genome is a singlemessenger-sense RNA. The term “messenger-sense” denotes that the viralRNAs can be directly translated to yield viral proteins, without theneed for an intervening transcription step.

[0006] Complete cDNA copies of the genetic component of TMV have beencloned. Construction of a library of subgenomic cDNA clones of TMV hasbeen described in Dawson et al., Proc. Natl. Acad. Sci. USA 83:1832-1836(1986) and Ahlquist et al., Proc. Natl. Acad. Sci. USA 81:7066-7070(1984). Several examples of TMV transcription vectors are describedbelow. DNA from each of the TMV cDNA-containing plasmids can be cleaved.The linear DNA thus produced can be transcribed in vitro in a reactioncatalyzed by RNA polymerase. A T7 promoter in the transcription vectorallows RNA synthesis to initiate at the 5′ terminus of each TMVsequence, and transcription continues to the end of the DNA template.The 5′ terminus of tobacco mosaic virus (TMV) RNA, was identified asm.sup.7 G.sup.5′ ppp.sup.5′ Gp. Zimmem, D., Nucleic Acid Res.2:1189-1201 (1975). Keith, J. and fraenkel-Conrat, H. FEBS Lett.57:31-33 (1975). Ahlquist, U.S. Pat. No. 5,500,360, working with Bromemosaic virus, reported that when transcription is carried out in thepresence of a synthetic cap structure, m.sup.7 GpppG, as described byContreras, R., et al. Nucleic Acids Res. 10:6353, (1982), RNAtranscripts are produced with the same capped 5′ ends as authentic BMVRNAs. Ahlquist concluded that these RNAs are active messengers in invitro translation systems and direct production of proteins with thesame electrophoretic mobilities as those translated from authentic BMVRNAs. However, Ahlquist found that, “if the cap analog was omittedduring in vitro transcription, no infection was detected, even ifinoculum concentration was increased 20-fold.” Further, Ahlquist taughtonly a viral vector having “no extraneous nonviral sequences between thecap and the 5′ terminus of the viral sequence.” In Ahlquist's work onBMV, U.S. Pat. No. 5,500,360, a transcription vector was employed whichpreserved the exact 5′ terminal nucleotide sequence of viral RNA. It isnow generally accepted that capping is necessary for infectivity andthat no intervening sequence can be present between the cap and the 5′ternminus of the viral sequence.

[0007] The work of Ahlquist leaves us with difficult problems toovercome if we are to obtain a workable viral vector or a commerciallyviable viral vector. One such problem is the cost of using cappingstructures and cap analogs. Another such problem is that multipartiteviral vectors are difficult to use relative to a single component viralvector. Multipartite viruses require more than one unit to infect andachieve replication in a host plant, and multipartite viruses require atrans acting replication element to achieve replication. No one has yetfound a way to unite the multiple strands of a multipartite virus intoan RNA molecule comprising the entire genome of a (+) strand RNA virusas suggested and claimed by Ahlquist.

[0008] Therefore, there is a need for a viral vector that can accept anintervening base or intervening sequence of bases between the cap andthe 5′ terminus of the viral sequence and undergo transcription andreplication. There is also a need for a viral vector that can undergotranscription and replication in the absence of a capping structure.

[0009] Here we teach solutions to the problem by demonstrating:

[0010] 1. Infection of a host and replication of a viral vector in vivoin the presence of a base or a sequence of bases placed 5′ to the originof replication in the absence of a capping structure or cap analog.

[0011] 2. Infection of a host and replication of a viral vector in vivoin the absence of a capping structure or a cap analog, and in theabsence of a base or a sequence of bases placed 5′ to the origin ofreplication.

[0012] 3. Infection of a host and replication of a viral vector in vivoin the presence of an intervening base or an intervening sequence ofbases placed 5′ to the origin of replication and in the presence of acapping structure or cap analog.

[0013] The viral vectors demonstrated here have utility in discovery thefunction of genes, and in production of therapeutic proteins.

SUMMARY OF THE INVENTION

[0014] The For the sake of brevity, the term “RNA virus” is used hereinto mean (+) strand replicating RNA viruses. Most single component RNAviruses have the advantage over multicomponent RNA viruses of having asingle RNA structure. Because they have a single RNA structure, thefunction of an exogenous RNA segment can be expressed in a host cell inthe absence of a trans-acting replication element. Further, the singlecomponent RNA virus does not express the 3a movement gene that isindigenous to the Brome mosaic virus.

[0015] The invention is based on the discovery that leaving out thecapping structure so that the virus transcript is uncapped can modifythe 5′ end of a single component RNA viral vector. The invention is alsobased on the discovery that the 5′ end of a single component RNA viralvector can be modified by inserting a base or a sequence of bases aheadof the 5′ terminus of the viral sequence. The invention is also based onthe discovery that the 5′ end of a single component RNA viral vector canbe modified by inserting an intervening base or an intervening sequenceof bases between the cap and the 5′ terminus of the viral sequence. Thegenome of a virus, modified in each of these three ways, can be furthermodified to include an exogenous RNA segment. The further modified RNAcan be introduced into a host cell where it will replicate and expressthe exogenous RNA segment. The recipient cell is thereby phenotypicallytransformed and may contribute to a genotypically transformed organism,as well.

[0016] Phenotypically transformed plants and plant cells can be modifiedin vivo, in planta, in tissue culture, in cell culture or in the form ofprotoplasts. The exemplified embodiment of the invention is useful forproducing phenotypically transformed plants under field or greenhousegrowth conditions. Traits desirable for introduction in this mannerinclude, but are not limited to, pest resistance, pathogen resistance,herbicide tolerance or resistance, modified growth habit and modifiedmetabolic characteristics, such as the production of commercially usefulpeptides or pharmaceuticals in plants. The modifications can be appliedat any time during the growth cycle, depending on the need for thetrait. For example, resistance to a pest could be conferred only if thecrop were at risk for that pest, and at the time when the crop was mostlikely to be affected by the pest. Other traits can be used to enhancesecondary properties, for example to increase the protein content ofpost-harvest forage. Any plant variety susceptible to infection by asingle component RNA virus can be phenotypically transformed. The choiceof virus and the details of modification will be matters of choicedepending on parameters known and understood by those of ordinary skillin the art. Other uses for cells and organisms phenotypically orgenotypically modified by means of a modified RNA derived from an RNAvirus will be readily apparent to those skilled in the art, given a widerange of RNA viruses to modify and a wide range of susceptible host celltypes. Other uses for transformed animal cells, bacterial cells and thelike can be readily envisioned but are not demonstrated here.

[0017] Generally, the steps of a process for phenotypically transforminga cell or organism are:

[0018] forming a full-length cDNA transcript of the RNA virus;

[0019] cloning the cDNA in a transcription vector;

[0020] modifying the cDNA by inserting a non-viral DNA segment in aregion able to tolerate such insertion without disrupting RNAreplication thereof;

[0021] transcribing the modified cDNA corresponding to the RNA componentof the single component virus;

[0022] infecting virus-susceptible protoplasts, cells, tissues or wholeplants with transcribed RNA, either in solution or encapsidated, of themodified RNA comprising messenger-sense RNA containing an exogenous RNAsegment.

[0023] From this point, the steps to be followed will vary, depending onthe type of material infected and the route of infection. Protoplasts,cells and tissues of plants can be propagated vegetatively, regeneratedto yield whole plants by means of any technique suitable to theparticular plant variety infected, and transplanted to the field. Wholeplants can be infected in situ. Infected plants and plant cells canproduce many copies per cell of the modified viral RNA containing theexogenous RNA segment. If desired and if suitably inserted, by means ofprinciples and processes known in the art, the exogenous RNA segment canbe caused to carry out a function within the cell. Such a function couldbe a coding function, translated within the cell to yield a desiredpeptide or protein, or it could be a regulatory function, increasing,decreasing, and turning on or off the expression of certain genes withinthe cell. In principle, any function, which a segment of RNA is capableof providing, can be expressed within the cell. The exogenous RNAsegment thus expressed confers a new phenotypic trait to the transformedorganism, plant, cells, protoplasts or tissues.

[0024] The invention is exemplified herein by the modification of TMVRNA to contain a structural gene encoding green fluorescent protein(GFP) and the phenotypic modification of Nicotiana plants andprotoplasts therewith, yielding plants and protoplasts synthesizing GFP.The data presented herein are believed to represent the first instanceof phenotypic modification of a cell by means of an RNA virus which isuncapped and which has no base at the 5′ end of the uncapped viralsequence.

[0025] The data presented herein are believed to represent the firstinstance of phenotypic modification of a cell by means of an RNA viruswhich is uncapped and which has a single base or a sequence of bases atthe 5′ end of an uncapped viral sequence. The data presented herein arebelieved to represent the first instance of phenotypic modification of acell by means of an RNA virus, which contains an intervening base, orintervening sequence of bases between the cap and the 5′ end of theviral sequence.

[0026] The present invention involves a capped RNA molecule capable ofreplication in the cytoplasm of a host plant cell located in a hostplant. It is also capable of local spread in a host plant, and alsocapable systemic spread in a host plant. The Capped RNA molecule of thepresent invention comprises:

[0027] a) a cis-acting viral replication element obtained from asingle-component (+) strand RNA plant virus, the plant virus capable ofreplication, local and systemic spread in a host plant;

[0028] b) a sequence of one to three intervening bases located between acap and the 5′ terminus of the viral sequence; and

[0029] c) an exogenous RNA segment capable of expressing its function ina host plant cell; the exogenous RNA segment being located in a regionof said capped RNA molecule able to tolerate said exogenous RNA segmentwithout disrupting replication of said capped RNA molecule, wherein saiduncapped RNA molecule is capable of replication in the absence of atrans-acting viral replication element in the host plant cell.

[0030] According to the present invention the exogenous RNA segmentcodes for a peptide or protein, and/or a structural protein. Theexogenous RNA can be antisense RNA, or regulatory RNA, or RNA havingcatalytic properties. The cis-acting viral replication element can bederived from a tobacco mosaic virus or another single stranded RNAvirus. The RNA molecule can be encapsidated with viral coat protein. Atypical host for such a virus is Nicotiana sp.

[0031] The vector of the present invention typically goes through acloning stage to form a DNA transcription vector. The DNA transcriptionvector comprises cDNA having one strand complementary to a capped RNAmolecule, the capped RNA molecule being capable of replication, localand systemic spread in a host plant cell, and comprising:

[0032] a) a cis-acting viral replication element derived from asingle-component (+) strand RNA plant virus, the plant virus capable ofreplication, local and systemic spread in the cytoplasm of a host plantcell, located a host plant; and

[0033] b) a sequence of one to three intervening bases located betweenthe cap and the 5′ terminus of the viral sequence; and

[0034] c) an exogenous RNA segment capable of expressing its function ina host plant cell in a region of said capped RNA molecule able totolerate said exogenous RNA segment without disrupting RNA replicationof said capped RNA molecule; and wherein said capped RNA molecule iscapable of replication in the absence of a trans-acting viralreplication element in host plant cells.

[0035] A DNA transcription vector of the present invention includepBTI1037, pBTI SBS60, pBTI SBS60-29, pBTI1056, pBTI1057, andpBTI1056-GTN28.

[0036] The present invention includes a method of modifying a host plantphenotypically or biochemically, which method, comprise:

[0037] i) introducing into a host plant cell which is located within thehost plant a capped RNA molecule capable of replication in the cytoplasmof a host plant cell located in a host plant, the capped RNA molecule,comprising:

[0038] a) a cis-acting viral replication element derived from asingle-component (+) strand RNA plant virus, the plant virus capable ofreplication in host plant cells;

[0039] b) a sequence of one to three intervening bases located betweenthe cap and the 5′ terminus of the viral sequence; and

[0040] c) an exogenous RNA segment segment capable of expressing itsfunction in a host cell, said exogenous RNA segment being located in aregion of said capped RNA molecule able to tolerate said exogenous RNAsegment without disrupting RNA replication of said capped RNA molecule,and wherein said capped RNA molecule is capable of replication in theabsence of a trans-acting viral replication element in host plant cells,whereby the exogenous RNA segment confers a detectable trait in the hostplant.

[0041] This invention is also represented by (+) strand RNA virus fromthe group consisting of Carlavirus, Closteroviridae, Luteoviridae,Potexvirus, Potyviridae, Tombusviridae, Tymovirus and Tobamovirus. Theseviruses might be engineered with a second subgenomic promoter to controlexpression of the exogenous RNA.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042]FIG. 1 shows the sequence of pBTI 30BGFPc3 (p1037) (SEQ ID NO:22).

[0043]FIG. 2 shows the sequence of pBTI SBS60 (SEQ ID NO: 23).

[0044]FIG. 3 shows the sequence of pBTI SBS60-29 (SEQ ID NO: 24).

[0045]FIG. 4 shows the sequence of pBTI1056 (SEQ ID NO: 25).

[0046]FIG. 5 shows the sequence of pBTI SBS5 (p1057) (SEQ ID NO: 26).

[0047]FIG. 6 shows the sequence of pBTI1056-GTN28 (SEQ ID NO: 27).

DETAILED DESCRIPTION OF THE INVENTION

[0048] In order to facilitate understanding of the invention, certainterms used throughout are herein defined.

[0049] Assembly as used herein means The stage of replication duringwhich all the structural components come together at one site in thecell and the basic structure of the virus particle is formed.

[0050] Base as used herein means adenine, thymidine, guanine, andcytosine, which in the form of a nucleotide can bond with other bases toform a nucleotide sequence. As used herein, a “base sequence” or a“sequence of bases” refers to a nucleotide sequence. The bases usedherein are DNA bases, because all base or base sequence manipulationsare performed on plasmid DNA prior to transcription. Base might be usedinterchangeably with “nucleotide”.

[0051] Cis-acting replication element as used herein means that portionof the RNA genome of an RNA virus, which must be present in cis, thatis, present as part of each viral strand as a necessary condition forreplication. Virus replication of a single component virus such as TMVhas only cis-acting replication elements in its RNA. The cis-actingreplication element is composed of one or more segments of viral RNA,which must be present on any RNA molecule that is to be replicated byRNA replication. The segment will most likely be the 5′ terminal portionof the viral RNA molecule, and may include other portions as well. As isdemonstrated herein, using the example of TMV, portions of an RNA virusmolecule may be modified without disrupting replication. A viral ornon-viral gene may be inserted under the control of an additionalsubgenomic promoter. A viral or non-viral gene may be fused to a viralcoat protein gene either under the control of the original subgenomicpromoter of the viral coat protein gene, or under the control of anadditional subgenomic promoter.

[0052] Derived from as used herein means the viral source of an RNAsegment, which comprises part of the modified RNA. For example, for themodified RNAs described herein, substantial portions thereof are derivedfrom TMV. The manner of deriving, whether by direct recombination at theRNA level, by transcription or by reverse transcription does not matterfor the purpose of the invention. It is contemplated that modificationsmay be made within the cis-acting replication element and elsewhere forexample to modify the rate or amount of replication that is obtained.

[0053] In the case of modified RNAs exemplified herein, a transcriptionvector was employed which, preserved the exact 5′ terminal nucleotidesequence of viral RNA, but a) left the capping structure off, or b) leftthe capping structure off and added a single base to the 5′ terminalnucleotide sequence of the viral cDNA, or c) left the capping structureoff and added a sequence of bases to the 5′ terminal nucleotide sequenceof the viral cDNA, or d) inserted a single intervening base between thecapping structure and the 5′ terminal nucleotide sequence of the viralcDNA, or e) inserted an intervening sequence of bases between the capand the 5′ terminal nucleotide sequence. The use of such a vector intranscribing viral RNA will be preferred if preservation of the exactnucleotide sequence at the 5′ end is desired. The use of such a vectorin transcribing viral RNA will be preferred if the objective is to onlyremove the cap without further objectives with respect to the 5′ end ofthe virus. An RNA segment which has been derived from a given sourcevirus may, but need not be, identical in sequence to that segment as itexists in the virus. It will be understood that a cis-acting replicatingelement derived from a given RNA virus may in some cases have minormodifications in the nucleotide sequence thereof without substantiallyinterfering with RNA replication.

[0054] Exogenous RNA segment as used herein means a segment of RNA to beinserted into the virus RNA to be modified, the source of the exogenousRNA segment being different from the RNA virus itself. The source may beanother virus, a living organism such as a plant, animal, bacteria,virus or fungus, the exogenous RNA may be a chemically synthesized RNAor it may be a combination of the foregoing. The exogenous RNA segmentmay provide any function that is appropriate and known to be provided byan RNA segment. Such functions include, but are not limited to, a codingfunction in which the RNA acts as a messenger RNA encoding a sequencewhich, translated by the host cell, results in synthesis of a peptide orprotein having useful or desired properties. The RNA segment may also bestructural, as for example in ribosomal RNA, it may be regulatory, asfor example with small nuclear RNAs or anti-sense RNA, or it may becatalytic. A particularly interesting function is provided by anti-senseRNA, sometimes termed (−) strand RNA, which is in fact a sequencecomplementary to another RNA sequence present in the target cell whichcan, through complementary base pairing, bind to and inhibit thefunction of the RNA in the target cell. An exogenous RNA segment can bea complete or partial coding sequence.

[0055] Various aspects of the stages outlined in the Summary section canbe modified as needed, depending upon specific aspects of the virusselected as the transforming agent and of the RNA segment to beinserted. For example, if the inserted gene is in the form ofmessenger-sense RNA to be directly translated by the transformed cell,the gene must be free of intervening, nontranslated sequences, such asintrons. On the other hand, the inserted gene need not be a naturallyoccurring gene, but it may be modified, it may be a composite of morethan one coding segment, or it may encode more than one protein.Combining insertions and deletions in order to control the total lengthor other properties of the modified RNA molecule may also modify theRNA. The inserted non-viral gene may be either prokaryotic or eukaryoticin origin as long as it is in a form, which can be directly translatedby the translation machinery of the recipient cell. Eukaryotic genescontaining introns within the coding sequence must therefore be insertedin the form of a cDNA copy of the eukaryotic messenger RNA encoding thegene. The inserted gene may contain its own translation start signals,for example, a ribosomal binding site and start (AUG) codon, or it maybe inserted in a manner which takes advantage of one or more of thesecomponents preexisting in the viral RNA to be modified. Certainstructural constraints must be observed to preserve correct translationof the inserted sequence, according to principles well understood in theart. For example, if it is intended that the exogenous coding segment becombined with an endogenous coding segment, the coding segment to beinserted must be inserted in reading frame phase therewith and in thesame translational direction.

[0056] Gene expression as used herein means An important stage of viralreplication at which virus genetic information is expressed: one of themajor control points in replication.

[0057] Genome replication as used herein means The stage of viralreplication at which the virus genome is copied to form new progenygenomes.

[0058] “Host plant cell located in a host plant” as used herein means ahost plant cell that is part of a host plant organism, host planttissue, or host plant organ.

[0059] Host as used herein means a host plant, host plant tissue or hostcell within a host plant. In this context, a host plant is amulticellular organism having leaf and stem, and may be said to haveplant cells, and plant tissues, plant organs or plant parts such asroot, stem leaf, flower or seed. A host is capable of being infected byand capable of replicating a nucleic acid such as a plant viral nucleicacid.

[0060] Infection as used herein means the ability of a virus to transferits nucleic acid to a host or introduce a viral nucleic acid into ahost, wherein the viral nucleic acid is replicated, viral proteins aresynthesized, and new viral particles assembled. In this context, theterms “transmissible” and “infective” are used interchangeably herein.The term is also meant to include the ability of a selected nucleic acidsequence to integrate into a genome, chromosome or gene of a targetorganism.

[0061] Infect, Infecting or Infection as used herein mean the ability ofa virus to transfer its nucleic acid to a host or introduce a viralnucleic acid into a host, wherein the viral nucleic acid is replicated,viral proteins are synthesized, and new viral particles assembled.

[0062] Local Spread as used herein means the ability of a virus or viralvector to move through the plasmodesmata from an infected cell to anadjacent cell. By this method, a virus that is replicating within a cellwill spread copies of itself to several surrounding cells. Replicationof virus in the adjacent cells will usually result in another round oflocal spread of virus to further adjacent cells.

[0063] mRNA as used herein means Messenger RNA, translated on ribosomesto produce proteins.

[0064] Non-viral as used herein means any RNA segment, which is notnormally contained within the virus whose modification is exploited foreffecting gene transfer and is therefore used synonymously with“exogenous”. Therefore, a gene derived from a different virus speciesthan that modified is included within the meaning of the terms“non-viral” and “exogenous” for the purposes of describing theinvention. For example, a non-viral gene as the term is used hereincould include a gene derived from a bacterial virus, an animal virus, ora plant virus of a type distinguishable from the virus modified toeffect transformation. In addition, a non-viral gene may be a structuralgene derived from any prokaryotic or eukaryotic organism. It will beunderstood by those ordinarily skilled in the art that there may existcertain genes whose transfer does not result in obvious phenotypicmodification of the host cell. A phenotypic modification may occur, forexample, if the translation product of the non-viral gene is toxic tothe host cell, is degraded or processed in a manner, which renders itnon-functional or possesses structural features, which render itimpossible for the host cell to translate in sufficient quantities toconfer a detectable phenotype on the transformed cells. However, theinvention does not depend upon any specific property of an RNA segmentor gene being transferred. Therefore, the possible existence of RNAsegments or genes which fail to confer a readily observable phenotypictrait on recipient cells or plants is irrelevant to the invention and inany case will be readily recognizable by those of ordinary skill in theart without undue experimentation.

[0065] Phenotypic Trait as used herein means an observable, measurableor detectable property resulting from the expression or suppression of agene or genes. Phenotype includes both easily observable traits andbiochemical processes.

[0066] Plant Cell as used herein means the structural and physiologicalunit of plants, consisting of a protoplast and the cell wall.

[0067] Plant host as used herein means a cell, tissue or organismcapable of replicating a nucleic acid such as a plant viral nucleic acidand which is capable of being infected by a virus containing the viralvector or viral nucleic acid. As used herein, plant host is intended toinclude whole plant, plant cell, and plant tissues, plant organ or plantpart such as root, stem leaf, flower or seed.

[0068] Plant Organ as used herein means a distinct and visiblydifferentiated part of a plant, such as root, stem, leaf or embryo.

[0069] Plant Tissue as used herein means Any tissue of a plant in plantaor in culture. This term is intended to include a whole plant, plantcell, plant organ, protoplast, cell culture, or any group of plant cellsorganized into a structural and functional unit.

[0070] (+)sense RNA (plus-sense RNA) as used herein means A virus with asingle-stranded RNA genome of the same polarity (‘sense’) as mRNA.

[0071] Positive-sense Inhibition as used herein means A type of generegulation based on inhibition of gene expression believed to be due tothe presence in a cell of an RNA molecule substantially homologous to atleast a portion of the mRNA being translated. The RNA molecule can be anexogenous coding sequence carried by an RNA viral vector of the typediscussed herein.

[0072] Promoter as used herein means The 5′-flanking, non-codingsequence substantially adjacent a coding sequence which is involved inthe initiation of transcription of the coding sequence.

[0073] Protoplast as used herein means an isolated plant cell withoutsome or all of its cell wall.

[0074] Replication as used herein means Nucleic acid synthesis, theprocess of copying.

[0075] RNA virus as used herein means a virus whose genome is RNA insingle-stranded form, the single strand being a (+) strand, ormessenger-sense strand. Replication of the viral (+) strand in avirus-infected cell occurs by a process of direct RNA replication and istherefore distinguishable from the replication mechanism ofretroviruses, which undergo an intermediate step of reversetranscription in the host cell.

[0076] Systemic Spread as used herein means the ability of a virus tospread from the vicinity of an initial infection to distant leaves.Systemic spread is accomplished by entry of the virus into the fluidcirculation of the plant. From the point of entry, the virus is carrieddown to the roots and then up to other leaves.

[0077] Single component virus as used herein means a virus having asingle nucleic acid sequence; unipartite. The single component virus iscontrasted with the multicomponent virus, which has more than onenucleic acid component. Each component of a multicomponent virus isindividually encapsidated, separate from the other(s).

[0078] Subgenomic Promoter as used herein means A promoter of asubgenomic mRNA of a viral nucleic acid. Plant viral nucleic acid can bemodified to contain an exogenous nucleic acid sequence under the controlof a subgenomic promoter.

[0079] Vector as used herein means an organism responsible fortransmitting a pathogen from one host to another, e.g. a mosquito. (Inmolecular biology, a molecule used to clone nucleic acid sequences).

[0080] Viral Vector as used herein means a self-replicating RNA or DNAmolecule derived from a virus which transfers an RNA or DNA segmentbetween cells, such as bacteria, yeast, plant, or animal cells andcontains an exogenous DNA or RNA segment to be expressed in the host.

[0081] Trans-acting replication element—In contrast to the singlecomponent (unipartite) virus, replication of a multipartite virus suchas BMV depends upon the existence of one or more trans (diffusible)replication elements, which interact with a cis-acting replicationelement to carry out RNA replication. While trans-acting elements arenecessary for replication of a multipartite virus such as BMV, they neednot be present or coded for on the modified RNA provided they are madeavailable within the infected cell by some other means. For example, inthe case of a multipartite RNA virus, the trans-acting functions may beprovided by other, unmodified components of the viral genome used totransform the cells simultaneously with the modified RNA. The targetcell may also be modified in a previous step to provide constitutiveexpression of the trans-acting functions. In the case of a multipartitevirus, the cis-acting element is therefore defined in functional terms:any modification which destroys the ability of the RNA to replicate in acell known to contain the requisite trans-acting elements, is deemed tobe a modification in the cis-acting replication element. Conversely, anymodification, such as an insertion in a sequence region, which is ableto tolerate such insertion without disrupting replication, is amodification outside the cis-acting replication element. At one time, itwas commonly believed that all viruses contained a trans-actingreplication factor. Today we know that many, and possibly all singlecomponent viruses do not have a trans-acting replication element.

[0082] A first embodiment demonstrates a capped viral vector having asingle base inserted at the 5′ terminus of the viral sequence.

[0083] Another embodiment demonstrates a capped viral vector having asequence of bases inserted at the 5′ terminus of the viral sequence.

[0084] In another embodiment, a host cell is infected by a capped viralvector which has a single base inserted at the 5′ terminus of the viralsequence. The capped viral vector is able to infect, to reproduce, tosystemically infect the host plant, and to express an exogenous RNAsegment.

[0085] In another embodiment, a host cell is infected by a capped viralvector having a sequence of bases inserted at the 5′ terminus of theviral sequence. The capped viral vector is able to infect the host cell,to reproduce, to systemically infect the host plant, and to express anexogenous RNA segment.

[0086] Another embodiment demonstrates an uncapped viral vector.

[0087] In another embodiment, an uncapped viral vector infects a hostcell. The uncapped viral vector is able to reproduce, to systemicallyinfect the host and to express an exogenous RNA segment.

[0088] Another embodiment demonstrates an uncapped viral vector having asingle base inserted at the 5′ terminus of the viral sequence.

[0089] In another embodiment, an uncapped viral vector having a singlebase inserted at the 5′ terminus of the viral sequence infects a hostcell. The uncapped viral vector is able to reproduce, to systemicallyinfect the host and to express an exogenous RNA segment.

[0090] Another embodiment demonstrates an uncapped viral vector having asequence of bases inserted at the 5′ terminus of the viral sequence.

[0091] In another embodiment, a host cell is infected by an uncappedviral vector having a sequence of bases inserted at the 5′ terminus ofthe viral sequence. The uncapped viral vector is able to reproduce, tosystemically infect the host and to express an exogenous RNA segment.

[0092] An exogenous RNA segment may be inserted at any convenientinsertion site provided the insertion does not disrupt a sequenceessential for replication of the RNA within the host cell. For example,Dual Heterologous Subgenomic Promoter Expression System (DHSPES) in aplus stranded RNA vector has two subgenomic promoters. An exogenous RNAsegment can be expressed in this system by inserting the exogenous geneat the 3′ end of one of the subgenomic promoters. This system isdescribed in U.S. Pat. Nos. 5,316,931, 5,811,653, 5,589,367 and5,866,785, the disclosure of which is incorporated by reference. Anexogenous RNA segment under the control of a subgenomic promoter will beexpressed in the host plant. Each heterologous subgenomic promoter iscapable of transcribing or expressing adjacent genes or nucleic acidsequences in the plant host and incapable of recombination with eachother and with native subgenomic promoters. One or more non-nativenucleic acids may be inserted adjacent to the native plant viralsubgenomic promoter or the native and non-native plant viral subgenomicpromoters if more than one nucleic acid sequence is included. Moreover,it is specifically contemplated that two or more heterologous non-nativesubgenomic promoters may be used. The exogenous RNA segment may betranscribed or expressed in the host plant under the control of thesubgenomic promoter to produce the products of the exogenous RNAsegment.

[0093] A virus, whose coat protein is not essential for replication, anexogenous RNA segment may be inserted within or substituted for theregion, which normally codes for coat protein. As desired, regions whichcontribute to undesirable host cell responses may be deleted orinactivated, provided such changes do not adversely effect the abilityof the RNA to be replicated in the host cell. For many single componentviruses, a reduction in the rate of normal RNA replication is tolerableand will in some instances be preferred, since the amount of RNAproduced in a normal infection is more than enough to saturate theribosomes of the transformed cell.

[0094] The transformation process itself can be carried out by any meanswhereby RNA can be introduced into cells, whole plants, plant tissues orprotoplasts. The RNA alone or encapsidated in a virus particle caninfect host cells, except that the modified viral RNA containing anon-viral RNA segment is substituted for its counterpart in a normalinfection. Any other suitable means for introducing RNA into targetcells such as microinjection may be used. Other variables of theinfection process, such as pretreatment of the recipients, use ofencapsidated or unencapsidated RNA, are matters of choice which those ofordinary skill in the art will be able to manipulate to achieve desiredtransformation efficiency in a given situation. For instance, the choiceof single component plant RNA virus to be modified to achieve geneexpression in a given plant variety will depend upon known host rangeproperties of single component plant RNA viruses. For example, TMVinfects a variety of Nicotiana species and their related domesticatedrelatives.

[0095] Plant cells, which are infected in culture, will normally remaintransformed as the cells grow and divide since the RNA components areable to replicate and thus become distributed to daughter cells uponcell division. Plants regenerated from phenotypically modified cells,tissues or protoplasts remain phenotypically modified. Similarly, plantstransformed as seedlings remain transformed during growth. Timing ofapplication of the transforming components will be governed by theresult that is intended and by variations in susceptibility to thetransforming virus or viral RNA during various stages of plant growth.

[0096] Using the various embodiments of the invention, an exogenoussegment RNA sequence can be expressed in a host by adapting theinvention to any of a variety of embodiments set forth below forexpressing an exogenous RNA segment. In one embodiment, an exogenous RNAsegment is introduced into a plant host by way of a viral nucleic acidwhich comprises a native plant viral subgenomic promoter, a plant viralcoat protein coding sequence, and at least one exogenous RNA segmentunder the control of a non-native subgenomic promoter.

[0097] In another embodiment, plant viral nucleic acid sequences used inthe method of the present invention are characterized by the deletion ofthe native coat protein coding sequence in favor of a non-native plantviral coat protein coding sequence for the purpose of increasing hostrange. A non-native promoter, which could be the subgenomic promoter ofthe non-native coat protein coding sequence, controls expression of thenon-native coat protein coding sequence. The non-native coat proteincoding sequence is capable of expression in the plant host, of packagingthe recombinant plant viral nucleic acid, and ensuring a systemicinfection of a permissive host by the recombinant plant viral nucleicacid. The recombinant plant viral nucleic acid may contain one or moreadditional native or non-native subgenomic promoters.

[0098] In another embodiment, plant viral nucleic acids are used in thepresent invention wherein the native coat protein coding sequence isplaced adjacent to a non-native subgenomic promoter.

[0099] In another embodiment, plant viral nucleic acids are used in thepresent invention wherein the native coat protein gene is adjacent itssubgenomic promoter and one or more non-native subgenomic promoters havebeen inserted into the viral nucleic acid. The inserted non-nativesubgenomic promoters are capable of transcribing or expressing adjacentgenes in a plant host and are incapable of recombination with each otherand with native subgenomic promoters. Non-native nucleic acid sequencesmay be inserted adjacent the non-native subgenomic plant viral promoterssuch that the sequences are transcribed or expressed in the host plantunder control of the subgenomic promoters to produce the product of thenon-native nucleic acid. Alternatively, a non-native coat protein codingsequence may replace the native coat protein coding sequence.

[0100] In another embodiment, a viral expression vector contains anexogenous RNA segment encoding a polyprotein. At least one protein ofthe polyprotein is non-native to the vector. The non-native protein isreleased from the polyprotein by proteolytic processing catalyzed by atleast one protease in the polyprotein. The vector comprises: a) at leastone promoter; b) cDNA having a sequence, which codes for at least onepolyprotein from a polyprotein-producing virus; c) at least one uniquerestriction site flanking a 3′ terminus of the cDNA; and a cloningvehicle.

[0101] Additional embodiments use a viral expression vector encoding atleast one protein non-native to the vector that is released from atleast one polyprotein expressed by the vector by proteolytic processing.The vector comprises at least one promoter, cDNA having a sequence whichcodes for at least one polyprotein from a polyprotein-producing virus,may contain at least one restriction site flanking a 3′ terminus of saidcDNA and a cloning vehicle. Preferred embodiments include using apotyvirus as the polyprotein-producing virus, and especially preferredembodiments may use TEV (tobacco etch virus). A more detaileddescription of such vectors useful according to the method of thepresent invention may be found in U.S. Pat. Nos. 5,491,076 and 5,766,885to James C. Carrington et al., which are incorporated herein byreference.

[0102] In yet other embodiments, recombinant plant viruses are used,which encode for the expression of a fusion between a plant viral coatprotein and the amino acid product of the exogenous RNA of interest.Such a recombinant plant virus provides for a high level of expressionof a nucleic acid of interest. The location or locations where the viralcoat protein is joined to the amino acid product of the nucleic acid ofinterest may be referred to as the fusion joint. A given product of sucha construct may have one or more fusion joints. The fusion joint may belocated at the carboxyl terminus of the viral coat protein or the fusionjoint may be located at the amino terminus of the coat protein portionof the construct. In instances where the nucleic acid of interest islocated internal with respect to the 5′ and 3′ residues of the nucleicacid sequence encoding for the viral coat protein, there are two fusionjoints. That is, the nucleic acid of interest may be located 5′, 3′,upstream, downstream or within the coat protein. In some embodiments ofsuch recombinant plant viruses, a “leaky” start or stop codon may occurat a fusion joint, which sometimes does not result in translationaltermination. A more detailed description of some recombinant plantviruses according to this embodiment of the invention may be found inU.S. Pat. No. 5,977,438, the disclosure of which is incorporated hereinby reference.

[0103] In another embodiment an exogenous gene can be introduced intothe site of the coat protein gene of Potato virus X. Alternatively, anexogenous gene can be added to the PVX genome by partial duplication ofthe viral genome, so that expression of the exogenous gene is under thecontrol of the same promoter sequence that controls production of thecoat protein gene. Chapman, S. et al., The Plant Journal (1992) 2(4):549-557.

[0104] Those skilled in the art will understand that these embodimentsare representative only of many constructs, which may be useful toproduce localized or systemic expression of nucleic acids in hostorganisms such as plants. All such constructs are contemplated andintended to be within the scope of the present invention.

[0105] The following examples illustrate the principles of the inventionas applied to modification of TMV and the use of modified TMV containinga gene coding for green fluorescent protein (GFP) in the phenotypictransformation of Nicotiana plants and protoplasts. The followingexamples utilize many techniques well known and accessible to thoseskilled in the arts of molecular biology, cloning, plant cell biology,plant virology and plant tissue culture. Such methods are fullydescribed in one or more of the cited references if not described indetail herein. Unless specified otherwise, enzymes were obtained fromcommercial sources and were used according to the vendor'srecommendations or other variations known to the art. Those in the artalso know reagents, buffers and culture conditions and reactionconditions for various enzyme-catalyzed reactions. Reference workscontaining such standard techniques include the following: R. Wu, ed.(1979) Meth. Enzymol. 68; R. Wu et al., eds. (1983) Meth. Enzymol. 100,101; L. Grossman and K. Moldave, eds. (1980) Meth. Enzymol. 65; J. H.Miller (1972) Experiment's in Molecular Genetics; R. Davis et al. (1980)Advanced Bacterial Genetics; R. F. Schleif and P. C. Wensink (1982)Practical Methods in Molecular Biology; and T. Maniatis et al. (1982)Molecular Cloning.

[0106] Textual use of the name of a restriction endonuclease inisolation, e.g., “EcoRV” or “SphI” refers to use of that enzyme in anenzymatic digestion, except in a diagram where it can refer to the siteof a sequence susceptible to action of that enzyme, e.g., a restrictionsite. In the text, restriction sites are indicated by the additional useof the word “site”, e.g., “EcoRV site”. The additional use of the word“fragment”, indicates a linear double-stranded DNA molecule having endsgenerated by action of the named enzyme (e.g., a restriction fragment).A phrase such as, “EcoRV/SphI” “fragment” indicates that the restrictionfragment was generated by the action of two different enzymes, hereEcoRV and SphI, the two ends resulting from the action of differentenzymes. Note that the ends will have the characteristics of beingeither sticky (i.e., having a single strand of protrusion capable ofbase pairing with a complementary single-stranded oligonucleotide) orblunt (i.e., having no single-stranded protrusion). The specificity of asticky end will be determined by the sequence of nucleotides comprisingthe single-stranded protrusion, which in turn is determined by thespecificity of the enzyme, which produces it.

[0107] All plasmids are designated by a sequence of letters and numbersprefaced by a lower case “p”, for example, pBTI1037, pBTI1056, pBTI1057,pBTI SBS60, pBTI SBS60-29, or pBTI1056-GTN 28. Certain steps of cloning,selection and vector increase employed strains of E. Coli. While thestrains used herein have been designated, there are many equivalentstrains, available to the public that may be employed. The use of aparticular microorganism as a substitute for a strain designated hereinis a matter of routine choice available to those of ordinary skill inthe art, according to well-known principles.

EXAMPLES Example 1 Infectivity of Uncapped and Capped Transcripts

[0108] This example demonstrates the production of highly infectiousviral vector transcripts containing 5′ nucleotides with reference to thevirus vector.

[0109] Insertion of base or base sequence at the 5′ end of the TMV cDNA.

[0110] Nucleotides were added between the transcriptional start site forin vitro transcription, in this case the T7 promoter, and the start ofthe cDNA of TMV in order to maximize transcription product yield andpossibly obviate the need to cap virus transcripts to insureinfectivity. The relevant sequence is the T7 promoter indicated inshorthand as TATA, followed by the transcription start site “G”,followed by TATTTT . . . , which is the continuation of the cDNA of TMV.These are put together as . . . TATAG TATTT . . . (SEQ ID NO: 1). Thebase preceding the “ ” is the start site for transcription of the cDNA.The bolded letter is the first base followed by TATTTT . . . of the TMVcDNA. Three approaches were taken:

[0111] 1) addition of G, GG or GGG between the start site oftranscription and the first base of the cDNA (as in . . . TATAG GTATTTT. . . and associated sequences); (SEQ ID NO:1)   I. starting point...TATAG TATTTT... (SEQ ID NO:2)  II. addition of G ...TATAG GTATTTT...(SEQ ID NO:3) III. addition of GG ...TATAG GGTATTTT... (SEQ ID NO:4) IV. addition of GGG ...TATAG GGGTATTTT...

[0112] 2) addition of G and a random base (GN). As used herein, [N=A, T,C, or G]. VI represents addition of two random bases (N2). VIIrepresents a G and two random bases (GNN). VIII represents three randombases (N3) between the start site of transcription and the TMV cDNA.(SEQ ID NO:5)    V. addition of GN ...TATAG NGTATTTT... (SEQ ID NO:6)  VI. addition of N2 ...TATAGN NNTATTTT... (SEQ ID NO:7)  VII. additionof GNN ...TATAG NNGTATTTT... (SEQ ID NO:8) VIII. addition of NNN...TATAGN NNTATTTT... (SEQ ID NO:9)   IX. addition of GNG ...TATAGGNGTATTT...

[0113] 3) addition of a GT and a single random base (GTN) between thestart site of transcription and the TMV cDNA (. . . TATAG TNGTATTTT, SEQID NO: 10 . . . and associated sequences).    X. addition of GTN...TATAG TNGTATTTT... (SEQ ID NO:11)   XI. addition of GTC, ...TATAGTCGTATTTT... (SEQ ID NO:12)  XII. addition of (GTN).sub.2 ...TATAGTNGTNGTATTTT... (SEQ ID NO:13) XIII. addition of (GTN).sub.4 ...TATAGTNGTNGTNGTNGTATTTT... (SEQ ID NO:14)  XIV. addition of GTATTT ...TATAGTATTTGTATTTT,... (SEQ ID NO:15)

[0114] The use of random bases was based on the hypothesis that aparticular base may be best suited for an additional nucleotide attachedto the cDNA, since it will be complementary to the normal non-templatedbase incorporated at the 3′-end of the TMV (−) strand RNA. This allowsfor more ready mis-initiation and restoration of wild type sequence. TheGTN would allow the mimicking of two potential sites for initiation, theadded and the native sequence, and facilitate more ready mis-initiationof transcription in vivo to restore the native TMV cDNA sequence.Approaches included cloning GFP expressing TMV vector sequences intovectors containing:

[0115] 1) an extra G,

[0116] 2) an extra GG or

[0117] 3) an extra GGG bases using standard molecular biologytechniques.

[0118] Likewise, full length PCR of TMV expression clone 1056 was doneto add

[0119] 4) N2,

[0120] 5) N3 and

[0121] 6) GTN bases between the T7 promoter and the TMV cDNA.

[0122] Construction of Plasmid

[0123] DNA oligonucleotide primers were synthesized to contain a 5′EcoRV site, an entire T7 RNA polymerase promoter, any extra nucleotides,and the 5′-terminal 20 bases of the TMV cDNA. These primers contain inthe position for extra nucleotides, either none for constructs withsequence . . . TATAG TATTT . . . , a “G” for constructs with sequence .. . TATAG GTATTT . . . , a “GN” for constructs with sequence . . . TATAGNGTATTT . . . or a “GTN” for constructs with sequence . . . TATAGTNGTATTT . . . , where indicates the base preceding is the start sitefor transcription.

[0124] Examples of 5′ primers used to construct variant TMV constructs:5′ GGCGATATCTAATACGACTCACTATAGTNGTAT (SEQ ID NO:16) TTTTACAACAATTACC5′ GGCGATATCTAATACGACTCACTATAGNGTATT (SEQ ID NO:17) TTTACAACAATTACC5′ GGCGATATCTAATACGACTCACTATAGNNGTAT (SEQ ID NO:18) TTTTACAACAATTACC5′ GCGATATCTAATACGACTCACTATAGNNNGTAT (SEQ ID NO:19) TTTTACAACAATTACC5′ GGCGATATCTAATACGACTCACTATAGTNGTNG (SEQ ID NO:20) TATTTTTACAACAATTAC

[0125] GATATC is the EcoRV restriction enzyme recognition site.Underlined is the T7 RNA polymerase promoter. The added bases betweenthe T7 promoter and the TMV cDNA are in bold. The 5′ 20 bases of TMVcDNA are shown following the added bases.

[0126] We used the following 3′-primer, which anneals to TMV nucleotides1034 to 1056:

[0127] 5′ CACTATCTACACTTTTATGGGCC (SEQ ID NO: 21).

[0128] These 5′ primers and a 3′ primer containing sequences in the TMVcDNA surrounding the SphI site at position 445 were used to amplify aportion of the TMV cDNA (.about.500 bp in length) by the polymerasechain reaction (PCR). The PCR products were purified by agarose gelelectrophoresis and standard gel extraction procedures and digested withEcoRV and SphI. The DNA fragments were then ligated into a plasmiddigested with EcoRV and SphI. The digestion removed the identicalportion of the genome and replaced it with the PCR fragment. DNAsequencing of the 5′ end of the TMV cDNA and T7 promoter junctionanalyzed by agarose gel electrophoresis and the recombinants. Theseplasmids were then used for in vitro transcription using T7 RNApolymerase.

[0129] In vitro Transcription

[0130] Several TMV-based virus expression vectors were initially used inthese studies. Vector pBTI 1056 contains the T7 promoter (underlined)followed directly by the virus cDNA sequence (. . . TATAGTATT . . .),and vector pBTI SBS60-29 contains the T7 promoter followed by an extraguanine residue, then by the virus cDNA sequence (. . . TATAGGTATT . ..). Both expression vectors express an exogenous cycle 3, shuffled greenfluorescent protein (GFPc3) in localized infection sites andsystemically infected tissue of infected plants.

[0131] Transcriptions of each plasmid were carried out in the absence ofcap analogue (uncapped) or in the presence of 8-fold greaterconcentration of RNA cap analogue than rGTP (capped). “r” meansribosomal.

[0132] Cap Transcriptions

[0133] 1.2 .mu.l 20 mM rATP, rCTP, rUTP, 2 mM rGTP solution

[0134] 2 .mu.l 10 mM RNA cap analogue (New England Biolabs catalog#1404, methylated cap analogue)

[0135] 1 .mu.l Rnase Inhibitor 20 U (Promega N2511)

[0136] 1 .mu.l T7 RNA polymerase 30 U (Ambion 2085)

[0137] 2 .mu.l T7 RNA polymerase buffer (Ambion n supplied with enzyme)

[0138] 0.5 mg of transcriptional plasmid DNA

[0139] Raise volume to 20 .mu.l

[0140] Incubate at 37.degree. C. for 1.5 hours

[0141] Analyze by agarose gel electrophoresis of 0.5 .mu.l solution.

[0142] Non-Cap Transcriptions

[0143] 1.2 .mu.l 20 mM rATP, rCTP, rUTP

[0144] 4.3 .mu.l 20 mM rGTP

[0145] 1 .mu.l Rnase Inhibitor 20 U (Promega N2511)

[0146] 1 .mu.l T7 RNA polymerase 30 U (Ambion 2085)

[0147] 2 .mu.l T7 RNA polymerase buffer (Ambion, supplied with enzyme)

[0148] 0.5 mg of transcriptional plasmid DNA

[0149] Raise volume to 20 .mu.l

[0150] Incubate at 37.degree. C. for 1.5 hours.

[0151] Analyze by agarose gel electrophoresis of 0.5 .mu.l solution.

[0152] There are other methods for transcription. This method is notintended to be limiting. The volume of rGTP is also not limiting. Othervolumes can be used. While methylated cap is used in these experiments,for purposes of this invention, unmethylated cap, New England Biolabscatalog #1407, may also be used if cap is desired.

[0153] Description of Vectors pBTI SBS5, pBTI 1056, pBTI SBS60, pBTISBS60-29, and pBTI 1056 GTN-28

[0154] Vector p30BGFPc3 is the base vector or starting point. Each clonecomparison is outlined below. pBTI SBS5, pSBS60 and p1056 are comparedwith p3OBGFPc3. P1056GTN-28 is compared with p1056 and pSBS60-29 scompared with pSBS60. “nt” means nucleotide. “aa” means amino acid. 1.pBTI SBS5 (pBTI 1057) SEQ DATA vs pBTI 30BGFPc3 (Pbti 1037) 8 nt changes4 aa changes nt 1138 pBTI SB S5 A to G mutation (E to G change of aa 357of 126K protein) nt 1268 T to C (silent) nt 2382 pBTI SBS5 A to Gmutation (K to E change of aa 772 of 126K protein) nt 3120 T to Cmutation (silent) nt 3632 pBTI SBS5 G to A mutation (silent) nt 5213 Cto T mutation (T to I change of aa 104 of 30K protein) nt 5303 pBTI SBS5A to G mutation (K to R change of aa 134 of 30K protein) nt 5896 C to Amutation (silent) 2. pBTI SBS60 SEQ DATA vs. pBTI 30BGFPc3 (pBTI 1037) 6nt changes 1 aa change nt 1268 T to C (silent) nt 3120 T to C mutation(silent) nt 4100 pBTI SBS60 T to C mutation (silent) nt 5213 C to Tmutation (T to I change of aa 104 of 30K protein, shared with pBTI SBS5)nt 5634 pBTI SBS60 A to G mutation (silent) nt 5896 C to A mutation(silent)

[0155] There is no nucleotide “nt” sequence inserted between the T7promoter sequence and the 5′ most base of the TMV U1 cDNA to form (. . .TATAGTATTTT . . .). In the short hand used herein . . . TATA representsthe T7 promoter, there is no base or sequence of bases inserted betweenthe T7 promoter and the GTATTTT . . . represents the 5′ most bases ofthe TMV U1 cDNA. 3. pBTI 1056 SEQ DATA vs. pBTI 30BGFPc3 (pBTI 1037) 2nt changes 2 aa change nt 5213 C to T mutation (T to I change of aa 104of 30k) nt 5402 G to A mutation (R to K change of aa 167 of 30k)

[0156] There is no nt sequence inserted between the T7 promoter sequenceand the 5′ most base of the TMV U1 cDNA to form (. . . TATAGTATTTT . ..). In the short hand used herein . . . TATA represents the T7 promoter,there is no base or sequence of bases inserted between the T7 promoterand the GTATTTT . . . represents the 5′ most bases of the TMV U1 cDNA.pBTI1056.

[0157] 4. pBTI 1056 GTN-28 SEQ DATA vs. pBTI 1056 nt sequence is GTCinserted between the T7 promoter sequence and the 5′ most base of theTMV U1 cDNA to form (. . . TATAGTCGTATTTT . . .). In the short hand usedherein . . . TATA represents the T7 promoter, GTC is the insertedsequence of nucleotides, and GTATTTT . . . represents the 5′ most basesof the TMV U1 cDNA

[0158] 5. pBTI SBS 60-29 SEQ DATA vs. pBTI SBS60 nt G is insertedbetween the T7 promoter sequence and the 5′ most base of the TMV U1 cDNAto form (. . . TATAGGTATTTT . . .). In the short hand used herein . . .TATA represents the T7 promoter, G is the inserted nucleotide, andGTATTTT . . . represents the 5′ most bases of the TMV U1 cDNA.

[0159] Table 1 summarizes the vectors and host plants used in thefollowing experiments; the nucleotide sequence of each vector whichcontains the T7 promoter and the start of the cDNA of TMV is listed inthe Table. TABLE 1 Viral Cap Host Foreign Plant+, Vector 5′ nucleotidesequence −Plant Gene tissue pBTI 1056 TATAGTATTTT + and NB and GFPc3leaf - NB 30K pBTI SBS60- TATAGGTATTTT + and NB and GFPc3 leaf - 29 NB30K pBTI SBS60 TATAGTATTTT + and NB GFPc3 protoplasts pBTI 1056-TATAGTCGTATTTT + and NB and GFPc3 leaf - GTN28 NB 30K

[0160] Data of Cap and Non-cap Transcriptions of pBTI1056 and PBTISBS60-29.

[0161] Nicotiana tabacum plants were infected with either capped oruncapped transcriptions (as described above) of pBTI 1056 and pBTISBS60-29. Transcriptions were mixed with abrasive and inoculated onexpanded older leaves of a wild type Nicotiana benthamiana (Nb) plantand a Nb plant expressing a TMV U1 30 k movement protein transgene (Nb30K). Four days post inoculation (dpi), long wave UV light was used tojudge the number of infection sites on the inoculated leaves of theplants. Systemic, noninoculated tissues were monitored from 4 dpi on forappearance of systemic infection indicating vascular movement of theinoculated virus. Table 2 shows the results of one representativeexperiment. An extra G, . . . TATAG GTATTTT . . . is found to be welltolerated as an additional 5′ nucleotide on the 5′ end of TMV vector RNAtranscripts. Both capped and uncapped transcripts are infectious. Extraguanine residues located between the T7 promoter and the first base of avirus cDNA as demonstrated by pBTISBS60-29 lead to an increased amountof RNA transcript. TABLE 2 Local Systemic infection sites InfectionConstruct Nb Nb 30K Nb Nb 30K pBTI 1056 Capped 5 6 yes yes Uncapped 0 5no yes pBTI SBS60-29 Capped 6 6 yes yes Uncapped 1 5 yes yes

[0162] Results of Cap and Non-cap Transcriptions of pBTI SBS60

[0163] Nicotiana tabacum protoplasts were infected with either capped oruncapped transcriptions (as described above) of pBTI SBS60 whichcontains the T7 promoter followed directly by the virus cDNA sequence(TATAGTATT . . .). This expression vector also expresses the GFPc3 genein infected cells and tissues. Nicotiana tabacum protoplasts weretransfected with 1 l of each transcription. Approximately 36 hours postinfection transfected protoplasts were viewed under UV illumination andcells showing GFPc3 expression. Approximately 80% of cells transfectedwith the capped pBTI SBS60 transcripts showed GFP expression while 5% ofcells transfected with uncapped transcripts showed GFP expression. Theseexperiments were repeated with higher amounts of uncapped inoculum. Inthis case a higher proportion of cells, >30% were found to be infectedat this time with uncapped transcripts, where >90% of cells infectedwith greater amounts of capped transcripts were scored infected.

[0164] Data of Cap and Non-cap Transcriptions of pBTI1056 GTN-28

[0165] TMV-based virus expression vector pBTI 1056 GTN-28 contains theT7 promoter (underlined) followed GTC bases (bold) then the virus cDNAsequence (. . . TATAGTCGTATT, SEQ ID NO: 10, . . .). This expressionvector expresses the exogenous cycle 3 shuffled green fluorescentprotein (GFPc3) in localized infection sites and systemically infectedtissue of infected plants. This vector was transcribed in vitro in thepresence (capped) and absence (uncapped) of cap analogue as describedabove. Transcriptions were mixed with abrasive and inoculated onexpanded older leaves of a wild type Nicotiana benthamiana (Nb) plantand a Nb plant expressing a TMV U1 30k movement protein transgene (Nb30K). Four days post inoculation (dpi) long wave UV light was used tojudge the number of infection sites on the inoculated leaves of theplants. Systemic, non-inoculated tissues were monitored from 4 dpi onfor appearance of systemic infection indicating vascular movement of theinoculated virus. Table 3 shows data from two representative experimentsat 11 dpi. TABLE 3 Local Systemic infection sites Infection Construct NbNb 30K Nb Nb 30K Experiment 1 pBTI 1056 GTN-28 Capped 18 25 yes yesUncapped 2 4 yes yes Experiment 2 pBTI 1056 GTN-28 Capped 8 12 yes yesUncapped 3 7 yes yes

[0166] Extra GTN such as GTC residues located between the T7 promoterand the first base of a virus cDNA (pBTI 1056 GTN-28) lead to increasedamount of RNA transcript as predicted by previous work with phagepolymerases. These polymerases tend to initiate more efficiently at . .. TATAGTNG or . . . TATAGTCG than . . . TATAG. This has an indirecteffect on the relative infectivity of uncapped transcripts in thatgreater amounts are synthesized per reaction resulting in enhancedinfectivity.

[0167] Discussion and Conclusions

[0168] The foregoing examples demonstrate that, contrary to thepracticed art in scientific literature and in issued patents (Ahlquistet al., U.S. Pat. No. 5,500,360), uncapped transcripts for virusexpression vectors are infective in both whole plants and in plantcells, however with much lower specific infectivity. Therefore, cappingis not a prerequisite for establishing an infection of a virusexpression vector in plants; capping just increases the efficiency ofinfection. This reduced efficiency can be overcome, to some extent, byproviding excess in vitro transcription product in an infection reactionfor plants or plant cells. These data further support the claimsconcerning the utility of uncapped transcripts to initiate infections byplant virus expression vectors and further demonstrates that theintroduction of extra, non-viral nucleotides at the 5′-end of in vitrotranscripts does not preclude infectivity of uncapped transcripts. Weconclude that while many similarities between plant viruses can becited, there are specific differences between the Brome mosaic virus andthe Tobamovirus group, which provide specific advantages to using asingle-component Tobamovirus-derived vector. The results also show thatreduced efficiency can be overcome, to some extent, by using atransgenic host plant or transgenic host plant cell, which expresses oneor more RNA binding viral proteins. The expression of the 30K movementprotein of TMV in transgenic plants also has the unexpected effect ofequalizing the relative specific infectivity of uncapped verses cappedtranscripts. The mechanism behind this effect is not fully understood.

[0169] Further modifications and improvements following and embodyingthe teachings and disclosures herein are deemed to be within the scopeof the invention, as set forth in the appended claims.

[0170] Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications could be made without departing from the spirit of theinvention. Further modifications and improvements following andembodying the teachings and disclosures herein are deemed to be withinthe scope of the invention, as set forth in the appended claims. Allpatent applications, publications and other documents cited herein areincorporated by reference in their entirety for all purposes.

1 27 1 11 DNA Tobacco mosaic virus 1 tatagtattt t 11 2 12 DNA Tobaccomosaic virus 2 tataggtatt tt 12 3 13 DNA Tobacco mosaic virus 3tatagggtat ttt 13 4 14 DNA Tobacco mosaic virus 4 tataggggta tttt 14 513 DNA Tobacco mosaic virus misc_feature n= A, T, C or G 5 tatagngtatttt 13 6 14 DNA Tobacco mosaic virus misc_feature N= A, T, C or G 6tatagnnnta tttt 14 7 10 DNA Tobacco mosaic virus misc_feature (6)..(7)N= A, T, C or G 7 tatagnngta 10 8 14 DNA Tobacco mosaic virusmisc_feature (6)..(8) N= A, T, C or G 8 tatagnnnta tttt 14 9 13 DNATobacco mosaic virus misc_feature (7)..(7) N= A, T, C or G 9 tataggngtattt 13 10 14 DNA Tobacco mosaic virus misc_feature (7)..(7) N= A, T, Cor G 10 tatagtngta tttt 14 11 14 DNA Tobacco mosaic virus misc_feature(7)..(7) N= A, T, C or G 11 tatagtngta tttt 14 12 14 DNA Tobacco mosaicvirus 12 tatagtcgta tttt 14 13 17 DNA Tobacco mosaic virus misc_feature(7)..(7) N= A, T, C or G 13 tatagtngtn gtatttt 17 14 23 DNA Tobaccomosaic virus misc_feature (7)..(7) N= A, T, C or G 14 tatagtngtngtngtngtat ttt 23 15 17 DNA Tobacco mosaic virus 15 tatagtattt gtatttt17 16 49 DNA Tobacco mosaic virus misc_feature (29)..(29) N= A, T, C orG 16 ggcgatatct aatacgactc actatagtng tatttttaca acaattacc 49 17 48 DNATobacco mosaic virus misc_feature (28)..(28) N= A, T, C or G 17ggcgatatct aatacgactc actatagngt atttttacaa caattacc 48 18 49 DNATobacco mosaic virus misc_feature (28)..(28) N= A, T, C or G 18ggcgatatct aatacgactc actatagnng tatttttaca acaattacc 49 19 50 DNATobacco mosaic virus misc_feature (28)..(30) N= A, T, C or G 19ggcgatatct aatacgactc actatagnnn gtatttttac aacaattacc 50 20 50 DNATobacco mosaic virus misc_feature (29)..(29) N= A, T, C or G 20ggcgatatct aatacgactc actatagtng tngtattttt acaacaatta 50 21 23 DNATobacco mosaic virus 21 cactatctac acttttatgg gcc 23 22 7685 DNA Tobaccomosaic virus 22 gtatttttac aacaattacc aacaacaaca aacaacagac aacattacaattactattta 60 caattacaat ggcatacaca cagacagcta ccacatcagc tttgctggacactgtccgag 120 gaaacaactc cttggtcaat gatctagcaa agcgtcgtct ttacgacacagcggttgaag 180 agtttaacgc tcgtgaccgc aggcccaagg tgaacttttc aaaagtaataagcgaggagc 240 agacgcttat tgctacccgg gcgtatccag aattccaaat tacattttataacacgcaaa 300 atgccgtgca ttcgcttgca ggtggattgc gatctttaga actggaatatctgatgatgc 360 aaattcccta cggatcattg acttatgaca taggcgggaa ttttgcatcgcatctgttca 420 agggacgagc atatgtacac tgctgcatgc ccaacctgga cgttcgagacatcatgcggc 480 acgaaggcca gaaagacagt attgaactat acctttctag gctagagagaggggggaaaa 540 cagtccccaa cttccaaaag gaagcatttg acagatacgc agaaattcctgaagacgctg 600 tctgtcacaa tactttccag acatgcgaac atcagccgat gcagcaatcaggcagagtgt 660 atgccattgc gctacacagc atatatgaca taccagccga tgagttcggggcggcactct 720 tgaggaaaaa tgtccatacg tgctatgccg ctttccactt ctccgagaacctgcttcttg 780 aagattcatg cgtcaatttg gacgaaatca acgcgtgttt ttcgcgcgatggagacaagt 840 tgaccttttc ttttgcatca gagagtactc ttaattactg tcatagttattctaatattc 900 ttaagtatgt gtgcaaaact tacttcccgg cctctaatag agaggtttacatgaaggagt 960 ttttagtcac cagagttaat acctggtttt gtaagttttc tagaatagatacttttcttt 1020 tgtacaaagg tgtggcccat aaaagtgtag atagtgagca gttttatactgcaatggaag 1080 acgcatggca ttacaaaaag actcttgcaa tgtgcaacag cgagagaatcctccttgagg 1140 attcatcatc agtcaattac tggtttccca aaatgaggga tatggtcatcgtaccattat 1200 tcgacatttc tttggagact agtaagagga cgcgcaagga agtcttagtgtccaaggatt 1260 tcgtgtttac agtgcttaac cacattcgaa cataccaggc gaaagctcttacatacgcaa 1320 atgttttgtc cttcgtcgaa tcgattcgat cgagggtaat cattaacggtgtgacagcga 1380 ggtccgaatg ggatgtggac aaatctttgt tacaatcctt gtccatgacgttttacctgc 1440 atactaagct tgccgttcta aaggatgact tactgattag caagtttagtctcggttcga 1500 aaacggtgtg ccagcatgtg tgggatgaga tttcgctggc gtttgggaacgcatttccct 1560 ccgtgaaaga gaggctcttg aacaggaaac ttatcagagt ggcaggcgacgcattagaga 1620 tcagggtgcc tgatctatat gtgaccttcc acgacagatt agtgactgagtacaaggcct 1680 ctgtggacat gcctgcgctt gacattagga agaagatgga agaaacggaagtgatgtaca 1740 atgcactttc agaattatcg gtgttaaggg agtctgacaa attcgatgttgatgtttttt 1800 cccagatgtg ccaatctttg gaagttgacc caatgacggc agcgaaggttatagtcgcgg 1860 tcatgagcaa tgagagcggt ctgactctca catttgaacg acctactgaggcgaatgttg 1920 cgctagcttt acaggatcaa gagaaggctt cagaaggtgc attggtagttacctcaagag 1980 aagttgaaga accgtccatg aagggttcga tggccagagg agagttacaattagctggtc 2040 ttgctggaga tcatccggaa tcgtcctatt ctaagaacga ggagatagagtctttagagc 2100 agtttcatat ggcgacggca gattcgttaa ttcgtaagca gatgagctcgattgtgtaca 2160 cgggtccgat taaagttcag caaatgaaaa actttatcga tagcctggtagcatcactat 2220 ctgctgcggt gtcgaatctc gtcaagatcc tcaaagatac agctgctattgaccttgaaa 2280 cccgtcaaaa gtttggagtc ttggatgttg catctaggaa gtggttaatcaaaccaacgg 2340 ccaagagtca tgcatggggt gttgttgaaa cccacgcgag gaagtatcatgtggcgcttt 2400 tggaatatga tgagcagggt gtggtgacat gcgatgattg gagaagagtagctgttagct 2460 ctgagtctgt tgtttattcc gacatggcga aactcagaac tctgcgcagactgcttcgaa 2520 acggagaacc gcatgtcagt agcgcaaagg ttgttcttgt ggacggagttccgggctgtg 2580 gaaaaaccaa agaaattctt tccagggtta attttgatga agatctaattttagtacctg 2640 ggaagcaagc cgcggaaatg atcagaagac gtgcgaattc ctcagggattattgtggcca 2700 cgaaggacaa cgttaaaacc gttgattctt tcatgatgaa ttttgggaaaagcacacgct 2760 gtcagttcaa gaggttattc attgatgaag ggttgatgtt gcatactggttgtgttaatt 2820 ttcttgtggc gatgtcattg tgcgaaattg catatgttta cggagacacacagcagattc 2880 catacatcaa tagagtttca ggattcccgt accccgccca ttttgccaaattggaagttg 2940 acgaggtgga gacacgcaga actactctcc gttgtccagc cgatgtcacacattatctga 3000 acaggagata tgagggcttt gtcatgagca cttcttcggt taaaaagtctgtttcgcagg 3060 agatggtcgg cggagccgcc gtgatcaatc cgatctcaaa acccttgcatggcaagatct 3120 tgacttttac ccaatcggat aaagaagctc tgctttcaag agggtattcagatgttcaca 3180 ctgtgcatga agtgcaaggc gagacatact ctgatgtttc actagttaggttaaccccta 3240 caccggtctc catcattgca ggagacagcc cacatgtttt ggtcgcattgtcaaggcaca 3300 cctgttcgct caagtactac actgttgtta tggatccttt agttagtatcattagagatc 3360 tagagaaact tagctcgtac ttgttagata tgtataaggt cgatgcaggaacacaatagc 3420 aattacagat tgactcggtg ttcaaaggtt ccaatctttt tgttgcagcgccaaagactg 3480 gtgatatttc tgatatgcag ttttactatg ataagtgtct cccaggcaacagcaccatga 3540 tgaataattt tgatgctgtt accatgaggt tgactgacat ttcattgaatgtcaaagatt 3600 gcatattgga tatgtctaag tctgttgctg cgcctaagga tcaaatcaaaccactaatac 3660 ctatggtacg aacggcggca gaaatgccac gccagactgg actattggaaaatttagtgg 3720 cgatgattaa aagaaacttt aacgcacccg agttgtctgg catcattgatattgaaaata 3780 ctgcatcttt ggttgtagat aagttttttg atagttattt gcttaaagaaaaaagaaaac 3840 caaataaaaa tgtttctttg ttcagtagag agtctctcaa tagatggttagaaaagcagg 3900 aacaggtaac aataggccag ctcgcagatt ttgattttgt ggatttgccagcagttgatc 3960 agtacagaca catgattaaa gcacaaccca aacaaaagtt ggacacttcaatccaaacgg 4020 agtacccggc tttgcagacg attgtgtacc attcaaaaaa gatcaatgcaatattcggcc 4080 cgttgtttag tgagcttact aggcaattac tggacagtgt tgattcgagcagatttttgt 4140 ttttcacaag aaagacacca gcgcagattg aggatttctt cggagatctcgacagtcatg 4200 tgccgatgga tgtcttggag ctggatatat caaaatacga caaatctcagaatgaattcc 4260 actgtgcagt agaatacgag atctggcgaa gattgggttt cgaagacttcttgggagaag 4320 tttggaaaca agggcataga aagaccaccc tcaaggatta taccgcaggtataaaaactt 4380 gcatctggta tcaaagaaag agcggggacg tcacgacgtt cattggaaacactgtgatca 4440 ttgctgcatg tttggcctcg atgcttccga tggagaaaat aatcaaaggagccttttgcg 4500 gtgacgatag tctgctgtac tttccaaagg gttgtgagtt tccggatgtgcaacactccg 4560 cgaatcttat gtggaatttt gaagcaaaac tgtttaaaaa acagtatggatacttttgcg 4620 gaagatatgt aatacatcac gacagaggat gcattgtgta ttacgatcccctaaagttga 4680 tctcgaaact tggtgctaaa cacatcaagg attgggaaca cttggaggagttcagaaggt 4740 ctctttgtga tgttgctgtt tcgttgaaca attgtgcgta ttacacacagttggacgacg 4800 ctgtatggga ggttcataag accgcccctc caggttcgtt tgtttataaaagtctggtga 4860 agtatttgtc tgataaagtt ctttttagaa gtttgtttat agatggctctagttgttaaa 4920 ggaaaagtga atatcaatga gtttatcgac ctgacaaaaa tggagaagatcttaccgtcg 4980 atgtttaccc ctgtaaagag tgttatgtgt tccaaagttg ataaaataatggttcatgag 5040 aatgagtcat tgtcaggggt gaaccttctt aaaggagtta agcttattgatagtggatac 5100 gtctgtttag ccggtttggt cgtcacgggc gagtggaact tgcctgacaattgcagagga 5160 ggtgtgagcg tgtgtctggt ggacaaaagg atggaaagag ccgacgaggccactctcgga 5220 tcttactaca cagcagctgc aaagaaaaga tttcagttca aggtcgttcccaattatgct 5280 ataaccaccc aggacgcgat gaaaaacgtc tggcaagttt tagttaatattagaaatgtg 5340 aagatgtcag cgggtttctg tccgctttct ctggagtttg tgtcggtgtgtattgtttat 5400 agaaataata taaaattagg tttgagagag aagattacaa acgtgagagacggagggccc 5460 atggaactta cagaagaagt cgttgatgag ttcatggaag atgtccctatgtcgatcagg 5520 cttgcaaagt ttcgatctcg aaccggaaaa aagagtgatg tccgcaaagggaaaaatagt 5580 agtagtgatc ggtcagtgcc gaacaagaac tatagaaatg ttaaggattttggaggaatg 5640 agttttaaaa agaataattt aatcgatgat gattcggagg ctactgtcgccgaatcggat 5700 tcgttttaaa tagatcttac agtatcacta ctccatctca gttcgtgttcttgtcattaa 5760 ttaaatggct agcaaaggag aagaactttt cactggagtt gtcccaattcttgttgaatt 5820 agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaaggtgatgctac 5880 atacggaaag cttaccctta aatttatttg cactactgga aaactacctgttccatggcc 5940 aacacttgtc actactttct cttatggtgt tcaatgcttt tcccgttatccggatcatat 6000 gaaacggcat gactttttca agagtgccat gcccgaaggt tatgtacaggaacgcactat 6060 atctttcaaa gatgacggga actacaagac gcgtgctgaa gtcaagtttgaaggtgatac 6120 ccttgttaat cgtatcgagt taaaaggtat tgattttaaa gaagatggaaacattctcgg 6180 acacaaactc gagtacaact ataactcaca caatgtatac atcacggcagacaaacaaaa 6240 gaatggaatc aaagctaact tcaaaattcg ccacaacatt gaagatggatccgttcaact 6300 agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttttaccagacaa 6360 ccattacctg tcgacacaat ctgccctttc gaaagatccc aacgaaaagcgtgaccacat 6420 gggccttctt gagtttgtaa ctgctgctgg gattacacat ggcatggatgagctctacaa 6480 ataatgacac tcgaggggta gtcaagatgc ataataaata acggattgtgtccgtaatca 6540 cacgtggtgc gtacgataac gcatagtgtt tttccctcca cttaaatcgaagggttgtgt 6600 cttggatcgc gcgggtcaaa tgtatatggt tcatatacat ccgcaggcacgtaataaagc 6660 gaggggttcg ggtcgaggtc ggctgtgaaa ctcgaaaagg ttccggaaaacaaaaaagag 6720 agtggtaggt aatagtgtta ataataagaa aataaataat agtggtaagaaaggtttgaa 6780 agttgaggaa attgaggata atgtaagtga tgacgagtct atcgcgtcatcgagtacgtt 6840 ttaatcaata tgccttatac aatcaactct ccgagccaat ttgtttacttaagttccgct 6900 tatgcagatc ctgtgcagct gatcaatctg tgtacaaatg cattgggtaaccagtttcaa 6960 acgcaacaag ctaggacaac agtccaacag caatttgcgg atgcctggaaacctgtgcct 7020 agtatgacag tgagatttcc tgcatcggat ttctatgtgt atagatataattcgacgctt 7080 gatccgttga tcacggcgtt attaaatagc ttcgatacta gaaatagaataatagaggtt 7140 gataatcaac ccgcaccgaa tactactgaa atcgttaacg cgactcagagggtagacgat 7200 gcgactgtag ctataagggc ttcaatcaat aatttggcta atgaactggttcgtggaact 7260 ggcatgttca atcaagcaag ctttgagact gctagtggac ttgtctggaccacaactccg 7320 gctacttagc tattgttgtg agatttccta aaataaagtc actgaagacttaaaattcag 7380 ggtggctgat accaaaatca gcagtggttg ttcgtccact taaatataacgattgtcata 7440 tctggatcca acagttaaac catgtgatgg tgtatactgt ggtatggcgtaaaacaacgg 7500 aaaagtcgct gaagacttaa aattcagggt ggctgatacc aaaatcagcagtggttgttc 7560 gtccacttaa aaataacgat tgtcatatct ggatccaaca gttaaaccatgtgatggtgt 7620 atactgtggt atggcgtaaa caacggagag gttcgaatcc tcccctaaccgcgggtagcg 7680 gccca 7685 23 7686 DNA Tobacco mosaic virus 23gtatttttac aacaattacc aacaacaaca aacaacagac aacattacaa ttactattta 60caattacaat ggcatacaca cagacagcta ccacatcagc tttgctggac actgtccgag 120gaaacaactc cttggtcaat gatctagcaa agcgtcgtct ttacgacaca gcggttgaag 180agtttaacgc tcgtgaccgc aggcccaagg tgaacttttc aaaagtaata agcgaggagc 240agacgcttat tgctacccgg gcgtatccag aattccaaat tacattttat aacacgcaaa 300atgccgtgca ttcgcttgca ggtggattgc gatctttaga actggaatat ctgatgatgc 360aaattcccta cggatcattg acttatgaca taggcgggaa ttttgcatcg catctgttca 420agggacgagc atatgtacac tgctgcatgc ccaacctgga cgttcgagac atcatgcggc 480acgaaggcca gaaagacagt attgaactat acctttctag gctagagaga ggggggaaaa 540cagtccccaa cttccaaaag gaagcatttg acagatacgc agaaattcct gaagacgctg 600tctgtcacaa tactttccag acatgcgaac atcagccgat gcagcaatca ggcagagtgt 660atgccattgc gctacacagc atatatgaca taccagccga tgagttcggg gcggcactct 720tgaggaaaaa tgtccatacg tgctatgccg ctttccactt ctccgagaac ctgcttcttg 780aagattcatg cgtcaatttg gacgaaatca acgcgtgttt ttcgcgcgat ggagacaagt 840tgaccttttc ttttgcatca gagagtactc ttaattactg tcatagttat tctaatattc 900ttaagtatgt gtgcaaaact tacttcccgg cctctaatag agaggtttac atgaaggagt 960ttttagtcac cagagttaat acctggtttt gtaagttttc tagaatagat acttttcttt 1020tgtacaaagg tgtggcccat aaaagtgtag atagtgagca gttttatact gcaatggaag 1080acgcatggca ttacaaaaag actcttgcaa tgtgcaacag cgagagaatc ctccttgagg 1140attcatcatc agtcaattac tggtttccca aaatgaggga tatggtcatc gtaccattat 1200tcgacatttc tttggagact agtaagagga cgcgcaagga agtcttagtg tccaaggatt 1260tcgtgttcac agtgcttaac cacattcgaa cataccaggc gaaagctctt acatacgcaa 1320atgttttgtc cttcgtcgaa tcgattcgat cgagggtaat cattaacggt gtgacagcga 1380ggtccgaatg ggatgtggac aaatctttgt tacaatcctt gtccatgacg ttttacctgc 1440atactaagct tgccgttcta aaggatgact tactgattag caagtttagt ctcggttcga 1500aaacggtgtg ccagcatgtg tgggatgaga tttcgctggc gtttgggaac gcatttccct 1560ccgtgaaaga gaggctcttg aacaggaaac ttatcagagt ggcaggcgac gcattagaga 1620tcagggtgcc tgatctatat gtgaccttcc acgacagatt agtgactgag tacaaggcct 1680ctgtggacat gcctgcgctt gacattagga agaagatgga agaaacggaa gtgatgtaca 1740atgcactttc agaattatcg gtgttaaggg agtctgacaa attcgatgtt gatgtttttt 1800cccagatgtg ccaatctttg gaagttgacc caatgacggc agcgaaggtt atagtcgcgg 1860tcatgagcaa tgagagcggt ctgactctca catttgaacg acctactgag gcgaatgttg 1920cgctagcttt acaggatcaa gagaaggctt cagaaggtgc attggtagtt acctcaagag 1980aagttgaaga accgtccatg aagggttcga tggccagagg agagttacaa ttagctggtc 2040ttgctggaga tcatccggaa tcgtcctatt ctaagaacga ggagatagag tctttagagc 2100agtttcatat ggcgacggca gattcgttaa ttcgtaagca gatgagctcg attgtgtaca 2160cgggtccgat taaagttcag caaatgaaaa actttatcga tagcctggta gcatcactat 2220ctgctgcggt gtcgaatctc gtcaagatcc tcaaagatac agctgctatt gaccttgaaa 2280cccgtcaaaa gtttggagtc ttggatgttg catctaggaa gtggttaatc aaaccaacgg 2340ccaagagtca tgcatggggt gttgttgaaa cccacgcgag gaagtatcat gtggcgcttt 2400tggaatatga tgagcagggt gtggtgacat gcgatgattg gagaagagta gctgttagct 2460ctgagtctgt tgtttattcc gacatggcga aactcagaac tctgcgcaga ctgcttcgaa 2520acggagaacc gcatgtcagt agcgcaaagg ttgttcttgt ggacggagtt ccgggctgtg 2580gaaaaaccaa agaaattctt tccagggtta attttgatga agatctaatt ttagtacctg 2640ggaagcaagc cgcggaaatg atcagaagac gtgcgaattc ctcagggatt attgtggcca 2700cgaaggacaa cgttaaaacc gttgattctt tcatgatgaa ttttgggaaa agcacacgct 2760gtcagttcaa gaggttattc attgatgaag ggttgatgtt gcatactggt tgtgttaatt 2820ttcttgtggc gatgtcattg tgcgaaattg catatgttta cggagacaca cagcagattc 2880catacatcaa tagagtttca ggattcccgt accccgccca ttttgccaaa ttggaagttg 2940acgaggtgga gacacgcaga actactctcc gttgtccagc cgatgtcaca cattatctga 3000acaggagata tgagggcttt gtcatgagca cttcttcggt taaaaagtct gtttcgcagg 3060agatggtcgg cggagccgcc gtgatcaatc cgatctcaaa acccttgcat ggcaagatcc 3120tgacttttac ccaatcggat aaagaagctc tgctttcaag agggtattca gatgttcaca 3180ctgtgcatga agtgcaaggc gagacatact ctgatgtttc actagttagg ttaaccccta 3240caccggtctc catcattgca ggagacagcc cacatgtttt ggtcgcattg tcaaggcaca 3300cctgttcgct caagtactac actgttgtta tggatccttt agttagtatc attagagatc 3360tagagaaact tagctcgtac ttgttagata tgtataaggt cgatgcagga acacaatagc 3420aattacagat tgactcggtg ttcaaaggtt ccaatctttt tgttgcagcg ccaaagactg 3480gtgatatttc tgatatgcag ttttactatg ataagtgtct cccaggcaac agcaccatga 3540tgaataattt tgatgctgtt accatgaggt tgactgacat ttcattgaat gtcaaagatt 3600gcatattgga tatgtctaag tctgttgctg cgcctaagga tcaaatcaaa ccactaatac 3660ctatggtacg aacggcggca gaaatgccac gccagactgg actattggaa aatttagtgg 3720cgatgattaa aagaaacttt aacgcacccg agttgtctgg catcattgat attgaaaata 3780ctgcatcttt ggttgtagat aagttttttg atagttattt gcttaaagaa aaaagaaaac 3840caaataaaaa tgtttctttg ttcagtagag agtctctcaa tagatggtta gaaaagcagg 3900aacaggtaac aataggccag ctcgcagatt ttgattttgt ggatttgcca gcagttgatc 3960agtacagaca catgattaaa gcacaaccca aacaaaagtt ggacacttca atccaaacgg 4020agtacccggc tttgcagacg attgtgtacc attcaaaaaa gatcaatgca atattcggcc 4080cgttgtttag tgagcttacc aggcaattac tggacagtgt tgattcgagc agatttttgt 4140ttttcacaag aaagacacca gcgcagattg aggatttctt cggagatctc gacagtcatg 4200tgccgatgga tgtcttggag ctggatatat caaaatacga caaatctcag aatgaattcc 4260actgtgcagt agaatacgag atctggcgaa gattgggttt cgaagacttc ttgggagaag 4320tttggaaaca agggcataga aagaccaccc tcaaggatta taccgcaggt ataaaaactt 4380gcatctggta tcaaagaaag agcggggacg tcacgacgtt cattggaaac actgtgatca 4440ttgctgcatg tttggcctcg atgcttccga tggagaaaat aatcaaagga gccttttgcg 4500gtgacgatag tctgctgtac tttccaaagg gttgtgagtt tccggatgtg caacactccg 4560cgaatcttat gtggaatttt gaagcaaaac tgtttaaaaa acagtatgga tacttttgcg 4620gaagatatgt aatacatcac gacagaggat gcattgtgta ttacgatccc ctaaagttga 4680tctcgaaact tggtgctaaa cacatcaagg attgggaaca cttggaggag ttcagaaggt 4740ctctttgtga tgttgctgtt tcgttgaaca attgtgcgta ttacacacag ttggacgacg 4800ctgtatggga ggttcataag accgcccctc caggttcgtt tgtttataaa agtctggtga 4860agtatttgtc tgataaagtt ctttttagaa gtttgtttat agatggctct agttgttaaa 4920ggaaaagtga atatcaatga gtttatcgac ctgacaaaaa tggagaagat cttaccgtcg 4980atgtttaccc ctgtaaagag tgttatgtgt tccaaagttg ataaaataat ggttcatgag 5040aatgagtcat tgtcaggggt gaaccttctt aaaggagtta agcttattga tagtggatac 5100gtctgtttag ccggtttggt cgtcacgggc gagtggaact tgcctgacaa ttgcagagga 5160ggtgtgagcg tgtgtctggt ggacaaaagg atggaaagag ccgacgaggc cattctcgga 5220tcttactaca cagcagctgc aaagaaaaga tttcagttca aggtcgttcc caattatgct 5280ataaccaccc aggacgcgat gaaaaacgtc tggcaagttt tagttaatat tagaaatgtg 5340aagatgtcag cgggtttctg tccgctttct ctggagtttg tgtcggtgtg tattgtttat 5400agaaataata taaaattagg tttgagagag aagattacaa acgtgagaga cggagggccc 5460atggaactta cagaagaagt cgttgatgag ttcatggaag atgtccctat gtcgatcagg 5520cttgcaaagt ttcgatctcg aaccggaaaa aagagtgatg tccgcaaagg gaaaaatagt 5580agtagtgatc ggtcagtgcc gaacaagaac tatagaaatg ttaaggattt tgggggaatg 5640agttttaaaa agaataattt aatcgatgat gattcggagg ctactgtcgc cgaatcggat 5700tcgttttaaa tagatcttac agtatcacta ctccatctca gttcgtgttc ttgtcattaa 5760ttaaatggct agcaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt 5820agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgctac 5880atacggaaag cttacactta aatttatttg cactactgga aaactacctg ttccatggcc 5940aacacttgtc actactttct cttatggtgt tcaatgcttt tcccgttatc cggatcatat 6000gaaacggcat gactttttca agagtgccat gcccgaaggt tatgtacagg aacgcactat 6060atctttcaaa gatgacggga actacaagac gcgtgctgaa gtcaagtttg aaggtgatac 6120ccttgttaat cgtatcgagt taaaaggtat tgattttaaa gaagatggaa acattctcgg 6180acacaaactc gagtacaact ataactcaca caatgtatac atcacggcag acaaacaaaa 6240gaatggaatc aaagctaact tcaaaattcg ccacaacatt gaagatggat ccgttcaact 6300agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa 6360ccattacctg tcgacacaat ctgccctttc gaaagatccc aacgaaaagc gtgaccacat 6420ggtccttctt gagtttgtaa ctgctgctgg gattacacat ggcatggatg agctctacaa 6480ataatgacac tcgaggggta gtcaagatgc ataataaata acggattgtg tccgtaatca 6540cacgtggtgc gtacgataac gcatagtgtt tttccctcca cttaaatcga agggttgtgt 6600cttggatcgc gcgggtcaaa tgtatatggt tcatatacat ccgcaggcac gtaataaagc 6660gaggggttcg ggtcgaggtc ggctgtgaaa ctcgaaaagg ttccggaaaa caaaaaagag 6720agtggtaggt aatagtgtta ataataagaa aataaataat agtggtaaga aaggtttgaa 6780agttgaggaa attgaggata atgtaagtga tgacgagtct atcgcgtcat cgagtacgtt 6840ttaatcaata tgccttatac aatcaactct ccgagccaat ttgtttactt aagttccgct 6900tatgcagatc ctgtgcagct gatcaatctg tgtacaaatg cattgggtaa ccagtttcaa 6960acgcaacaag ctaggacaac agtccaacag caatttgcgg atgcctggaa acctgtgcct 7020agtatgacag tgagatttcc tgcatcggat ttctatgtgt atagatataa ttcgacgctt 7080gatccgttga tcacggcgtt attaaatagc ttcgatacta gaaatagaat aatagaggtt 7140gataatcaac ccgcaccgaa tactactgaa atcgttaacg cgactcagag ggtagacgat 7200gcgactgtag ctataagggc ttcaatcaat aatttggcta atgaactggt tcgtggaact 7260ggcatgttca atcaagcaag ctttgagact gctagtggac ttgtctggac cacaactccg 7320gctacttagc tattgttgtg agatttccta aaataaagtc actgaagact taaaattcag 7380ggtggctgat accaaaatca gcagtggttg ttcgtccact taaatataac gattgtcata 7440tctggatcca acagttaaac catgtgatgg tgtatactgt ggtatggcgt aaaacaacgg 7500aaaagtcgct gaagacttaa aattcagggt ggctgatacc aaaatcagca gtggttgttc 7560gtccacttaa aaataacgat tgtcatatct ggatccaaca gttaaaccat gtgatggtgt 7620atactgtggt atggcgtaaa acaacggaga ggttcgaatc ctcccctaac cgcgggtagc 7680ggccca 7686 24 7687 DNA Tobacco mosaic virus 24 ggtattttta caacaattaccaacaacaac aaacaacaga caacattaca attactattt 60 acaattacaa tggcatacacacagacagct accacatcag ctttgctgga cactgtccga 120 ggaaacaact ccttggtcaatgatctagca aagcgtcgtc tttacgacac agcggttgaa 180 gagtttaacg ctcgtgaccgcaggcccaag gtgaactttt caaaagtaat aagcgaggag 240 cagacgctta ttgctacccgggcgtatcca gaattccaaa ttacatttta taacacgcaa 300 aatgccgtgc attcgcttgcaggtggattg cgatctttag aactggaata tctgatgatg 360 caaattccct acggatcattgacttatgac ataggcggga attttgcatc gcatctgttc 420 aagggacgag catatgtacactgctgcatg cccaacctgg acgttcgaga catcatgcgg 480 cacgaaggcc agaaagacagtattgaacta tacctttcta ggctagagag aggggggaaa 540 acagtcccca acttccaaaaggaagcattt gacagatacg cagaaattcc tgaagacgct 600 gtctgtcaca atactttccagacatgcgaa catcagccga tgcagcaatc aggcagagtg 660 tatgccattg cgctacacagcatatatgac ataccagccg atgagttcgg ggcggcactc 720 ttgaggaaaa atgtccatacgtgctatgcc gctttccact tctccgagaa cctgcttctt 780 gaagattcat gcgtcaatttggacgaaatc aacgcgtgtt tttcgcgcga tggagacaag 840 ttgacctttt cttttgcatcagagagtact cttaattact gtcatagtta ttctaatatt 900 cttaagtatg tgtgcaaaacttacttcccg gcctctaata gagaggttta catgaaggag 960 tttttagtca ccagagttaatacctggttt tgtaagtttt ctagaataga tacttttctt 1020 ttgtacaaag gtgtggcccataaaagtgta gatagtgagc agttttatac tgcaatggaa 1080 gacgcatggc attacaaaaagactcttgca atgtgcaaca gcgagagaat cctccttgag 1140 gattcatcat cagtcaattactggtttccc aaaatgaggg atatggtcat cgtaccatta 1200 ttcgacattt ctttggagactagtaagagg acgcgcaagg aagtcttagt gtccaaggat 1260 ttcgtgttca cagtgcttaaccacattcga acataccagg cgaaagctct tacatacgca 1320 aatgttttgt ccttcgtcgaatcgattcga tcgagggtaa tcattaacgg tgtgacagcg 1380 aggtccgaat gggatgtggacaaatctttg ttacaatcct tgtccatgac gttttacctg 1440 catactaagc ttgccgttctaaaggatgac ttactgatta gcaagtttag tctcggttcg 1500 aaaacggtgt gccagcatgtgtgggatgag atttcgctgg cgtttgggaa cgcatttccc 1560 tccgtgaaag agaggctcttgaacaggaaa cttatcagag tggcaggcga cgcattagag 1620 atcagggtgc ctgatctatatgtgaccttc cacgacagat tagtgactga gtacaaggcc 1680 tctgtggaca tgcctgcgcttgacattagg aagaagatgg aagaaacgga agtgatgtac 1740 aatgcacttt cagaattatcggtgttaagg gagtctgaca aattcgatgt tgatgttttt 1800 tcccagatgt gccaatctttggaagttgac ccaatgacgg cagcgaaggt tatagtcgcg 1860 gtcatgagca atgagagcggtctgactctc acatttgaac gacctactga ggcgaatgtt 1920 gcgctagctt tacaggatcaagagaaggct tcagaaggtg cattggtagt tacctcaaga 1980 gaagttgaag aaccgtccatgaagggttcg atggccagag gagagttaca attagctggt 2040 cttgctggag atcatccggaatcgtcctat tctaagaacg aggagataga gtctttagag 2100 cagtttcata tggcgacggcagattcgtta attcgtaagc agatgagctc gattgtgtac 2160 acgggtccga ttaaagttcagcaaatgaaa aactttatcg atagcctggt agcatcacta 2220 tctgctgcgg tgtcgaatctcgtcaagatc ctcaaagata cagctgctat tgaccttgaa 2280 acccgtcaaa agtttggagtcttggatgtt gcatctagga agtggttaat caaaccaacg 2340 gccaagagtc atgcatggggtgttgttgaa acccacgcga ggaagtatca tgtggcgctt 2400 ttggaatatg atgagcagggtgtggtgaca tgcgatgatt ggagaagagt agctgttagc 2460 tctgagtctg ttgtttattccgacatggcg aaactcagaa ctctgcgcag actgcttcga 2520 aacggagaac cgcatgtcagtagcgcaaag gttgttcttg tggacggagt tccgggctgt 2580 ggaaaaacca aagaaattctttccagggtt aattttgatg aagatctaat tttagtacct 2640 gggaagcaag ccgcggaaatgatcagaaga cgtgcgaatt cctcagggat tattgtggcc 2700 acgaaggaca acgttaaaaccgttgattct ttcatgatga attttgggaa aagcacacgc 2760 tgtcagttca agaggttattcattgatgaa gggttgatgt tgcatactgg ttgtgttaat 2820 tttcttgtgg cgatgtcattgtgcgaaatt gcatatgttt acggagacac acagcagatt 2880 ccatacatca atagagtttcaggattcccg taccccgccc attttgccaa attggaagtt 2940 gacgaggtgg agacacgcagaactactctc cgttgtccag ccgatgtcac acattatctg 3000 aacaggagat atgagggctttgtcatgagc acttcttcgg ttaaaaagtc tgtttcgcag 3060 gagatggtcg gcggagccgccgtgatcaat ccgatctcaa aacccttgca tggcaagatc 3120 ctgactttta cccaatcggataaagaagct ctgctttcaa gagggtattc agatgttcac 3180 actgtgcatg aagtgcaaggcgagacatac tctgatgttt cactagttag gttaacccct 3240 acaccggtct ccatcattgcaggagacagc ccacatgttt tggtcgcatt gtcaaggcac 3300 acctgttcgc tcaagtactacactgttgtt atggatcctt tagttagtat cattagagat 3360 ctagagaaac ttagctcgtacttgttagat atgtataagg tcgatgcagg aacacaatag 3420 caattacaga ttgactcggtgttcaaaggt tccaatcttt ttgttgcagc gccaaagact 3480 ggtgatattt ctgatatgcagttttactat gataagtgtc tcccaggcaa cagcaccatg 3540 atgaataatt ttgatgctgttaccatgagg ttgactgaca tttcattgaa tgtcaaagat 3600 tgcatattgg atatgtctaagtctgttgct gcgcctaagg atcaaatcaa accactaata 3660 cctatggtac gaacggcggcagaaatgcca cgccagactg gactattgga aaatttagtg 3720 gcgatgatta aaagaaactttaacgcaccc gagttgtctg gcatcattga tattgaaaat 3780 actgcatctt tggttgtagataagtttttt gatagttatt tgcttaaaga aaaaagaaaa 3840 ccaaataaaa atgtttctttgttcagtaga gagtctctca atagatggtt agaaaagcag 3900 gaacaggtaa caataggccagctcgcagat tttgattttg tggatttgcc agcagttgat 3960 cagtacagac acatgattaaagcacaaccc aaacaaaagt tggacacttc aatccaaacg 4020 gagtacccgg ctttgcagacgattgtgtac cattcaaaaa agatcaatgc aatattcggc 4080 ccgttgttta gtgagcttaccaggcaatta ctggacagtg ttgattcgag cagatttttg 4140 tttttcacaa gaaagacaccagcgcagatt gaggatttct tcggagatct cgacagtcat 4200 gtgccgatgg atgtcttggagctggatata tcaaaatacg acaaatctca gaatgaattc 4260 cactgtgcag tagaatacgagatctggcga agattgggtt tcgaagactt cttgggagaa 4320 gtttggaaac aagggcatagaaagaccacc ctcaaggatt ataccgcagg tataaaaact 4380 tgcatctggt atcaaagaaagagcggggac gtcacgacgt tcattggaaa cactgtgatc 4440 attgctgcat gtttggcctcgatgcttccg atggagaaaa taatcaaagg agccttttgc 4500 ggtgacgata gtctgctgtactttccaaag ggttgtgagt ttccggatgt gcaacactcc 4560 gcgaatctta tgtggaattttgaagcaaaa ctgtttaaaa aacagtatgg atacttttgc 4620 ggaagatatg taatacatcacgacagagga tgcattgtgt attacgatcc cctaaagttg 4680 atctcgaaac ttggtgctaaacacatcaag gattgggaac acttggagga gttcagaagg 4740 tctctttgtg atgttgctgtttcgttgaac aattgtgcgt attacacaca gttggacgac 4800 gctgtatggg aggttcataagaccgcccct ccaggttcgt ttgtttataa aagtctggtg 4860 aagtatttgt ctgataaagttctttttaga agtttgttta tagatggctc tagttgttaa 4920 aggaaaagtg aatatcaatgagtttatcga cctgacaaaa atggagaaga tcttaccgtc 4980 gatgtttacc cctgtaaagagtgttatgtg ttccaaagtt gataaaataa tggttcatga 5040 gaatgagtca ttgtcaggggtgaaccttct taaaggagtt aagcttattg atagtggata 5100 cgtctgttta gccggtttggtcgtcacggg cgagtggaac ttgcctgaca attgcagagg 5160 aggtgtgagc gtgtgtctggtggacaaaag gatggaaaga gccgacgagg ccattctcgg 5220 atcttactac acagcagctgcaaagaaaag atttcagttc aaggtcgttc ccaattatgc 5280 tataaccacc caggacgcgatgaaaaacgt ctggcaagtt ttagttaata ttagaaatgt 5340 gaagatgtca gcgggtttctgtccgctttc tctggagttt gtgtcggtgt gtattgttta 5400 tagaaataat ataaaattaggtttgagaga gaagattaca aacgtgagag acggagggcc 5460 catggaactt acagaagaagtcgttgatga gttcatggaa gatgtcccta tgtcgatcag 5520 gcttgcaaag tttcgatctcgaaccggaaa aaagagtgat gtccgcaaag ggaaaaatag 5580 tagtagtgat cggtcagtgccgaacaagaa ctatagaaat gttaaggatt ttgggggaat 5640 gagttttaaa aagaataatttaatcgatga tgattcggag gctactgtcg ccgaatcgga 5700 ttcgttttaa atagatcttacagtatcact actccatctc agttcgtgtt cttgtcatta 5760 attaaatggc tagcaaaggagaagaacttt tcactggagt tgtcccaatt cttgttgaat 5820 tagatggtga tgttaatgggcacaaatttt ctgtcagtgg agagggtgaa ggtgatgcta 5880 catacggaaa gcttacacttaaatttattt gcactactgg aaaactacct gttccatggc 5940 caacacttgt cactactttctcttatggtg ttcaatgctt ttcccgttat ccggatcata 6000 tgaaacggca tgactttttcaagagtgcca tgcccgaagg ttatgtacag gaacgcacta 6060 tatctttcaa agatgacgggaactacaaga cgcgtgctga agtcaagttt gaaggtgata 6120 cccttgttaa tcgtatcgagttaaaaggta ttgattttaa agaagatgga aacattctcg 6180 gacacaaact cgagtacaactataactcac acaatgtata catcacggca gacaaacaaa 6240 agaatggaat caaagctaacttcaaaattc gccacaacat tgaagatgga tccgttcaac 6300 tagcagacca ttatcaacaaaatactccaa ttggcgatgg ccctgtcctt ttaccagaca 6360 accattacct gtcgacacaatctgcccttt cgaaagatcc caacgaaaag cgtgaccaca 6420 tggtccttct tgagtttgtaactgctgctg ggattacaca tggcatggat gagctctaca 6480 aataatgaca ctcgaggggtagtcaagatg cataataaat aacggattgt gtccgtaatc 6540 acacgtggtg cgtacgataacgcatagtgt ttttccctcc acttaaatcg aagggttgtg 6600 tcttggatcg cgcgggtcaaatgtatatgg ttcatataca tccgcaggca cgtaataaag 6660 cgaggggttc gggtcgaggtcggctgtgaa actcgaaaag gttccggaaa acaaaaaaga 6720 gagtggtagg taatagtgttaataataaga aaataaataa tagtggtaag aaaggtttga 6780 aagttgagga aattgaggataatgtaagtg atgacgagtc tatcgcgtca tcgagtacgt 6840 tttaatcaat atgccttatacaatcaactc tccgagccaa tttgtttact taagttccgc 6900 ttatgcagat cctgtgcagctgatcaatct gtgtacaaat gcattgggta accagtttca 6960 aacgcaacaa gctaggacaacagtccaaca gcaatttgcg gatgcctgga aacctgtgcc 7020 tagtatgaca gtgagatttcctgcatcgga tttctatgtg tatagatata attcgacgct 7080 tgatccgttg atcacggcgttattaaatag cttcgatact agaaatagaa taatagaggt 7140 tgataatcaa cccgcaccgaatactactga aatcgttaac gcgactcaga gggtagacga 7200 tgcgactgta gctataagggcttcaatcaa taatttggct aatgaactgg ttcgtggaac 7260 tggcatgttc aatcaagcaagctttgagac tgctagtgga cttgtctgga ccacaactcc 7320 ggctacttag ctattgttgtgagatttcct aaaataaagt cactgaagac ttaaaattca 7380 gggtggctga taccaaaatcagcagtggtt gttcgtccac ttaaatataa cgattgtcat 7440 atctggatcc aacagttaaaccatgtgatg gtgtatactg tggtatggcg taaaacaacg 7500 gaaaagtcgc tgaagacttaaaattcaggg tggctgatac caaaatcagc agtggttgtt 7560 cgtccactta aaaataacgattgtcatatc tggatccaac agttaaacca tgtgatggtg 7620 tatactgtgg tatggcgtaaaacaacggag aggttcgaat cctcccctaa ccgcgggtag 7680 cggccca 7687 25 7685DNA Tobacco mosaic virus 25 gtatttttac aacaattacc aacaacaaca aacaacagacaacattacaa ttactattta 60 caattacaat ggcatacaca cagacagcta ccacatcagctttgctggac actgtccgag 120 gaaacaactc cttggtcaat gatctagcaa agcgtcgtctttacgacaca gcggttgaag 180 agtttaacgc tcgtgaccgc aggcccaagg tgaacttttcaaaagtaata agcgaggagc 240 agacgcttat tgctacccgg gcgtatccag aattccaaattacattttat aacacgcaaa 300 atgccgtgca ttcgcttgca ggtggattgc gatctttagaactggaatat ctgatgatgc 360 aaattcccta cggatcattg acttatgaca taggcgggaattttgcatcg catctgttca 420 agggacgagc atatgtacac tgctgcatgc ccaacctggacgttcgagac atcatgcggc 480 acgaaggcca gaaagacagt attgaactat acctttctaggctagagaga ggggggaaaa 540 cagtccccaa cttccaaaag gaagcatttg acagatacgcagaaattcct gaagacgctg 600 tctgtcacaa tactttccag acatgcgaac atcagccgatgcagcaatca ggcagagtgt 660 atgccattgc gctacacagc atatatgaca taccagccgatgagttcggg gcggcactct 720 tgaggaaaaa tgtccatacg tgctatgccg ctttccacttctccgagaac ctgcttcttg 780 aagattcatg cgtcaatttg gacgaaatca acgcgtgtttttcgcgcgat ggagacaagt 840 tgaccttttc ttttgcatca gagagtactc ttaattactgtcatagttat tctaatattc 900 ttaagtatgt gtgcaaaact tacttcccgg cctctaatagagaggtttac atgaaggagt 960 ttttagtcac cagagttaat acctggtttt gtaagttttctagaatagat acttttcttt 1020 tgtacaaagg tgtggcccat aaaagtgtag atagtgagcagttttatact gcaatggaag 1080 acgcatggca ttacaaaaag actcttgcaa tgtgcaacagcgagagaatc ctccttgagg 1140 attcatcatc agtcaattac tggtttccca aaatgagggatatggtcatc gtaccattat 1200 tcgacatttc tttggagact agtaagagga cgcgcaaggaagtcttagtg tccaaggatt 1260 tcgtgtttac agtgcttaac cacattcgaa cataccaggcgaaagctctt acatacgcaa 1320 atgttttgtc cttcgtcgaa tcgattcgat cgagggtaatcattaacggt gtgacagcga 1380 ggtccgaatg ggatgtggac aaatctttgt tacaatccttgtccatgacg ttttacctgc 1440 atactaagct tgccgttcta aaggatgact tactgattagcaagtttagt ctcggttcga 1500 aaacggtgtg ccagcatgtg tgggatgaga tttcgctggcgtttgggaac gcatttccct 1560 ccgtgaaaga gaggctcttg aacaggaaac ttatcagagtggcaggcgac gcattagaga 1620 tcagggtgcc tgatctatat gtgaccttcc acgacagattagtgactgag tacaaggcct 1680 ctgtggacat gcctgcgctt gacattagga agaagatggaagaaacggaa gtgatgtaca 1740 atgcactttc agaattatcg gtgttaaggg agtctgacaaattcgatgtt gatgtttttt 1800 cccagatgtg ccaatctttg gaagttgacc caatgacggcagcgaaggtt atagtcgcgg 1860 tcatgagcaa tgagagcggt ctgactctca catttgaacgacctactgag gcgaatgttg 1920 cgctagcttt acaggatcaa gagaaggctt cagaaggtgcattggtagtt acctcaagag 1980 aagttgaaga accgtccatg aagggttcga tggccagaggagagttacaa ttagctggtc 2040 ttgctggaga tcatccggaa tcgtcctatt ctaagaacgaggagatagag tctttagagc 2100 agtttcatat ggcgacggca gattcgttaa ttcgtaagcagatgagctcg attgtgtaca 2160 cgggtccgat taaagttcag caaatgaaaa actttatcgatagcctggta gcatcactat 2220 ctgctgcggt gtcgaatctc gtcaagatcc tcaaagatacagctgctatt gaccttgaaa 2280 cccgtcaaaa gtttggagtc ttggatgttg catctaggaagtggttaatc aaaccaacgg 2340 ccaagagtca tgcatggggt gttgttgaaa cccacgcgaggaagtatcat gtggcgcttt 2400 tggaatatga tgagcagggt gtggtgacat gcgatgattggagaagagta gctgttagct 2460 ctgagtctgt tgtttattcc gacatggcga aactcagaactctgcgcaga ctgcttcgaa 2520 acggagaacc gcatgtcagt agcgcaaagg ttgttcttgtggacggagtt ccgggctgtg 2580 gaaaaaccaa agaaattctt tccagggtta attttgatgaagatctaatt ttagtacctg 2640 ggaagcaagc cgcggaaatg atcagaagac gtgcgaattcctcagggatt attgtggcca 2700 cgaaggacaa cgttaaaacc gttgattctt tcatgatgaattttgggaaa agcacacgct 2760 gtcagttcaa gaggttattc attgatgaag ggttgatgttgcatactggt tgtgttaatt 2820 ttcttgtggc gatgtcattg tgcgaaattg catatgtttacggagacaca cagcagattc 2880 catacatcaa tagagtttca ggattcccgt accccgcccattttgccaaa ttggaagttg 2940 acgaggtgga gacacgcaga actactctcc gttgtccagccgatgtcaca cattatctga 3000 acaggagata tgagggcttt gtcatgagca cttcttcggttaaaaagtct gtttcgcagg 3060 agatggtcgg cggagccgcc gtgatcaatc cgatctcaaaacccttgcat ggcaagatct 3120 tgacttttac ccaatcggat aaagaagctc tgctttcaagagggtattca gatgttcaca 3180 ctgtgcatga agtgcaaggc gagacatact ctgatgtttcactagttagg ttaaccccta 3240 caccggtctc catcattgca ggagacagcc cacatgttttggtcgcattg tcaaggcaca 3300 cctgttcgct caagtactac actgttgtta tggatcctttagttagtatc attagagatc 3360 tagagaaact tagctcgtac ttgttagata tgtataaggtcgatgcagga acacaatagc 3420 aattacagat tgactcggtg ttcaaaggtt ccaatctttttgttgcagcg ccaaagactg 3480 gtgatatttc tgatatgcag ttttactatg ataagtgtctcccaggcaac agcaccatga 3540 tgaataattt tgatgctgtt accatgaggt tgactgacatttcattgaat gtcaaagatt 3600 gcatattgga tatgtctaag tctgttgctg cgcctaaggatcaaatcaaa ccactaatac 3660 ctatggtacg aacggcggca gaaatgccac gccagactggactattggaa aatttagtgg 3720 cgatgattaa aagaaacttt aacgcacccg agttgtctggcatcattgat attgaaaata 3780 ctgcatcttt ggttgtagat aagttttttg atagttatttgcttaaagaa aaaagaaaac 3840 caaataaaaa tgtttctttg ttcagtagag agtctctcaatagatggtta gaaaagcagg 3900 aacaggtaac aataggccag ctcgcagatt ttgattttgtggatttgcca gcagttgatc 3960 agtacagaca catgattaaa gcacaaccca aacaaaagttggacacttca atccaaacgg 4020 agtacccggc tttgcagacg attgtgtacc attcaaaaaagatcaatgca atattcggcc 4080 cgttgtttag tgagcttact aggcaattac tggacagtgttgattcgagc agatttttgt 4140 ttttcacaag aaagacacca gcgcagattg aggatttcttcggagatctc gacagtcatg 4200 tgccgatgga tgtcttggag ctggatatat caaaatacgacaaatctcag aatgaattcc 4260 actgtgcagt agaatacgag atctggcgaa gattgggtttcgaagacttc ttgggagaag 4320 tttggaaaca agggcataga aagaccaccc tcaaggattataccgcaggt ataaaaactt 4380 gcatctggta tcaaagaaag agcggggacg tcacgacgttcattggaaac actgtgatca 4440 ttgctgcatg tttggcctcg atgcttccga tggagaaaataatcaaagga gccttttgcg 4500 gtgacgatag tctgctgtac tttccaaagg gttgtgagtttccggatgtg caacactccg 4560 cgaatcttat gtggaatttt gaagcaaaac tgtttaaaaaacagtatgga tacttttgcg 4620 gaagatatgt aatacatcac gacagaggat gcattgtgtattacgatccc ctaaagttga 4680 tctcgaaact tggtgctaaa cacatcaagg attgggaacacttggaggag ttcagaaggt 4740 ctctttgtga tgttgctgtt tcgttgaaca attgtgcgtattacacacag ttggacgacg 4800 ctgtatggga ggttcataag accgcccctc caggttcgtttgtttataaa agtctggtga 4860 agtatttgtc tgataaagtt ctttttagaa gtttgtttatagatggctct agttgttaaa 4920 ggaaaagtga atatcaatga gtttatcgac ctgacaaaaatggagaagat cttaccgtcg 4980 atgtttaccc ctgtaaagag tgttatgtgt tccaaagttgataaaataat ggttcatgag 5040 aatgagtcat tgtcaggggt gaaccttctt aaaggagttaagcttattga tagtggatac 5100 gtctgtttag ccggtttggt cgtcacgggc gagtggaacttgcctgacaa ttgcagagga 5160 ggtgtgagcg tgtgtctggt ggacaaaagg atggaaagagccgacgaggc cattctcgga 5220 tcttactaca cagcagctgc aaagaaaaga tttcagttcaaggtcgttcc caattatgct 5280 ataaccaccc aggacgcgat gaaaaacgtc tggcaagttttagttaatat tagaaatgtg 5340 aagatgtcag cgggtttctg tccgctttct ctggagtttgtgtcggtgtg tattgtttat 5400 aaaaataata taaaattagg tttgagagag aagattacaaacgtgagaga cggagggccc 5460 atggaactta cagaagaagt cgttgatgag ttcatggaagatgtccctat gtcgatcagg 5520 cttgcaaagt ttcgatctcg aaccggaaaa aagagtgatgtccgcaaagg gaaaaatagt 5580 agtagtgatc ggtcagtgcc gaacaagaac tatagaaatgttaaggattt tggaggaatg 5640 agttttaaaa agaataattt aatcgatgat gattcggaggctactgtcgc cgaatcggat 5700 tcgttttaaa tagatcttac agtatcacta ctccatctcagttcgtgttc ttgtcattaa 5760 ttaaatggct agcaaaggag aagaactttt cactggagttgtcccaattc ttgttgaatt 5820 agatggtgat gttaatgggc acaaattttc tgtcagtggagagggtgaag gtgatgctac 5880 atacggaaag cttaccctta aatttatttg cactactggaaaactacctg ttccatggcc 5940 aacacttgtc actactttct cttatggtgt tcaatgcttttcccgttatc cggatcatat 6000 gaaacggcat gactttttca agagtgccat gcccgaaggttatgtacagg aacgcactat 6060 atctttcaaa gatgacggga actacaagac gcgtgctgaagtcaagtttg aaggtgatac 6120 ccttgttaat cgtatcgagt taaaaggtat tgattttaaagaagatggaa acattctcgg 6180 acacaaactc gagtacaact ataactcaca caatgtatacatcacggcag acaaacaaaa 6240 gaatggaatc aaagctaact tcaaaattcg ccacaacattgaagatggat ccgttcaact 6300 agcagaccat tatcaacaaa atactccaat tggcgatggccctgtccttt taccagacaa 6360 ccattacctg tcgacacaat ctgccctttc gaaagatcccaacgaaaagc gtgaccacat 6420 gggccttctt gagtttgtaa ctgctgctgg gattacacatggcatggatg agctctacaa 6480 ataatgacac tcgaggggta gtcaagatgc ataataaataacggattgtg tccgtaatca 6540 cacgtggtgc gtacgataac gcatagtgtt tttccctccacttaaatcga agggttgtgt 6600 cttggatcgc gcgggtcaaa tgtatatggt tcatatacatccgcaggcac gtaataaagc 6660 gaggggttcg ggtcgaggtc ggctgtgaaa ctcgaaaaggttccggaaaa caaaaaagag 6720 agtggtaggt aatagtgtta ataataagaa aataaataatagtggtaaga aaggtttgaa 6780 agttgaggaa attgaggata atgtaagtga tgacgagtctatcgcgtcat cgagtacgtt 6840 ttaatcaata tgccttatac aatcaactct ccgagccaatttgtttactt aagttccgct 6900 tatgcagatc ctgtgcagct gatcaatctg tgtacaaatgcattgggtaa ccagtttcaa 6960 acgcaacaag ctaggacaac agtccaacag caatttgcggatgcctggaa acctgtgcct 7020 agtatgacag tgagatttcc tgcatcggat ttctatgtgtatagatataa ttcgacgctt 7080 gatccgttga tcacggcgtt attaaatagc ttcgatactagaaatagaat aatagaggtt 7140 gataatcaac ccgcaccgaa tactactgaa atcgttaacgcgactcagag ggtagacgat 7200 gcgactgtag ctataagggc ttcaatcaat aatttggctaatgaactggt tcgtggaact 7260 ggcatgttca atcaagcaag ctttgagact gctagtggacttgtctggac cacaactccg 7320 gctacttagc tattgttgtg agatttccta aaataaagtcactgaagact taaaattcag 7380 ggtggctgat accaaaatca gcagtggttg ttcgtccacttaaatataac gattgtcata 7440 tctggatcca acagttaaac catgtgatgg tgtatactgtggtatggcgt aaaacaacgg 7500 aaaagtcgct gaagacttaa aattcagggt ggctgataccaaaatcagca gtggttgttc 7560 gtccacttaa aaataacgat tgtcatatct ggatccaacagttaaaccat gtgatggtgt 7620 atactgtggt atggcgtaaa caacggagag gttcgaatcctcccctaacc gcgggtagcg 7680 gccca 7685 26 7686 DNA Tobacco mosaic virus26 gtatttttac aacaattacc aacaacaaca aacaacagac aacattacaa ttactattta 60caattacaat ggcatacaca cagacagcta ccacatcagc tttgctggac actgtccgag 120gaaacaactc cttggtcaat gatctagcaa agcgtcgtct ttacgacaca gcggttgaag 180agtttaacgc tcgtgaccgc aggcccaagg tgaacttttc aaaagtaata agcgaggagc 240agacgcttat tgctacccgg gcgtatccag aattccaaat tacattttat aacacgcaaa 300atgccgtgca ttcgcttgca ggtggattgc gatctttaga actggaatat ctgatgatgc 360aaattcccta cggatcattg acttatgaca taggcgggaa ttttgcatcg catctgttca 420agggacgagc atatgtacac tgctgcatgc ccaacctgga cgttcgagac atcatgcggc 480acgaaggcca gaaagacagt attgaactat acctttctag gctagagaga ggggggaaaa 540cagtccccaa cttccaaaag gaagcatttg acagatacgc agaaattcct gaagacgctg 600tctgtcacaa tactttccag acatgcgaac atcagccgat gcagcaatca ggcagagtgt 660atgccattgc gctacacagc atatatgaca taccagccga tgagttcggg gcggcactct 720tgaggaaaaa tgtccatacg tgctatgccg ctttccactt ctccgagaac ctgcttcttg 780aagattcatg cgtcaatttg gacgaaatca acgcgtgttt ttcgcgcgat ggagacaagt 840tgaccttttc ttttgcatca gagagtactc ttaattactg tcatagttat tctaatattc 900ttaagtatgt gtgcaaaact tacttcccgg cctctaatag agaggtttac atgaaggagt 960ttttagtcac cagagttaat acctggtttt gtaagttttc tagaatagat acttttcttt 1020tgtacaaagg tgtggcccat aaaagtgtag atagtgagca gttttatact gcaatggaag 1080acgcatggca ttacaaaaag actcttgcaa tgtgcaacag cgagagaatc ctccttgggg 1140attcatcatc agtcaattac tggtttccca aaatgaggga tatggtcatc gtaccattat 1200tcgacatttc tttggagact agtaagagga cgcgcaagga agtcttagtg tccaaggatt 1260tcgtgttcac agtgcttaac cacattcgaa cataccaggc gaaagctctt acatacgcaa 1320atgttttgtc cttcgtcgaa tcgattcgat cgagggtaat cattaacggt gtgacagcga 1380ggtccgaatg ggatgtggac aaatctttgt tacaatcctt gtccatgacg ttttacctgc 1440atactaagct tgccgttcta aaggatgact tactgattag caagtttagt ctcggttcga 1500aaacggtgtg ccagcatgtg tgggatgaga tttcgctggc gtttgggaac gcatttccct 1560ccgtgaaaga gaggctcttg aacaggaaac ttatcagagt ggcaggcgac gcattagaga 1620tcagggtgcc tgatctatat gtgaccttcc acgacagatt agtgactgag tacaaggcct 1680ctgtggacat gcctgcgctt gacattagga agaagatgga agaaacggaa gtgatgtaca 1740atgcactttc agaattatcg gtgttaaggg agtctgacaa attcgatgtt gatgtttttt 1800cccagatgtg ccaatctttg gaagttgacc caatgacggc agcgaaggtt atagtcgcgg 1860tcatgagcaa tgagagcggt ctgactctca catttgaacg acctactgag gcgaatgttg 1920cgctagcttt acaggatcaa gagaaggctt cagaaggtgc attggtagtt acctcaagag 1980aagttgaaga accgtccatg aagggttcga tggccagagg agagttacaa ttagctggtc 2040ttgctggaga tcatccggaa tcgtcctatt ctaagaacga ggagatagag tctttagagc 2100agtttcatat ggcgacggca gattcgttaa ttcgtaagca gatgagctcg attgtgtaca 2160cgggtccgat taaagttcag caaatgaaaa actttatcga tagcctggta gcatcactat 2220ctgctgcggt gtcgaatctc gtcaagatcc tcaaagatac agctgctatt gaccttgaaa 2280cccgtcaaaa gtttggagtc ttggatgttg catctaggaa gtggttaatc aaaccaacgg 2340ccaagagtca tgcatggggt gttgttgaaa cccacgcgag ggagtatcat gtggcgcttt 2400tggaatatga tgagcagggt gtggtgacat gcgatgattg gagaagagta gctgttagct 2460ctgagtctgt tgtttattcc gacatggcga aactcagaac tctgcgcaga ctgcttcgaa 2520acggagaacc gcatgtcagt agcgcaaagg ttgttcttgt ggacggagtt ccgggctgtg 2580gaaaaaccaa agaaattctt tccagggtta attttgatga agatctaatt ttagtacctg 2640ggaagcaagc cgcggaaatg atcagaagac gtgcgaattc ctcagggatt attgtggcca 2700cgaaggacaa cgttaaaacc gttgattctt tcatgatgaa ttttgggaaa agcacacgct 2760gtcagttcaa gaggttattc attgatgaag ggttgatgtt gcatactggt tgtgttaatt 2820ttcttgtggc gatgtcattg tgcgaaattg catatgttta cggagacaca cagcagattc 2880catacatcaa tagagtttca ggattcccgt accccgccca ttttgccaaa ttggaagttg 2940acgaggtgga gacacgcaga actactctcc gttgtccagc cgatgtcaca cattatctga 3000acaggagata tgagggcttt gtcatgagca cttcttcggt taaaaagtct gtttcgcagg 3060agatggtcgg cggagccgcc gtgatcaatc cgatctcaaa acccttgcat ggcaagatcc 3120tgacttttac ccaatcggat aaagaagctc tgctttcaag agggtattca gatgttcaca 3180ctgtgcatga agtgcaaggc gagacatact ctgatgtttc actagttagg ttaaccccta 3240caccggtctc catcattgca ggagacagcc cacatgtttt ggtcgcattg tcaaggcaca 3300cctgttcgct caagtactac actgttgtta tggatccttt agttagtatc attagagatc 3360tagagaaact tagctcgtac ttgttagata tgtataaggt cgatgcagga acacaatagc 3420aattacagat tgactcggtg ttcaaaggtt ccaatctttt tgttgcagcg ccaaagactg 3480gtgatatttc tgatatgcag ttttactatg ataagtgtct cccaggcaac agcaccatga 3540tgaataattt tgatgctgtt accatgaggt tgactgacat ttcattgaat gtcaaagatt 3600gcatattgga tatgtctaag tctgttgctg cacctaagga tcaaatcaaa ccactaatac 3660ctatggtacg aacggcggca gaaatgccac gccagactgg actattggaa aatttagtgg 3720cgatgattaa aagaaacttt aacgcacccg agttgtctgg catcattgat attgaaaata 3780ctgcatcttt ggttgtagat aagttttttg atagttattt gcttaaagaa aaaagaaaac 3840caaataaaaa tgtttctttg ttcagtagag agtctctcaa tagatggtta gaaaagcagg 3900aacaggtaac aataggccag ctcgcagatt ttgattttgt ggatttgcca gcagttgatc 3960agtacagaca catgattaaa gcacaaccca aacaaaagtt ggacacttca atccaaacgg 4020agtacccggc tttgcagacg attgtgtacc attcaaaaaa gatcaatgca atattcggcc 4080cgttgtttag tgagcttact aggcaattac tggacagtgt tgattcgagc agatttttgt 4140ttttcacaag aaagacacca gcgcagattg aggatttctt cggagatctc gacagtcatg 4200tgccgatgga tgtcttggag ctggatatat caaaatacga caaatctcag aatgaattcc 4260actgtgcagt agaatacgag atctggcgaa gattgggttt cgaagacttc ttgggagaag 4320tttggaaaca agggcataga aagaccaccc tcaaggatta taccgcaggt ataaaaactt 4380gcatctggta tcaaagaaag agcggggacg tcacgacgtt cattggaaac actgtgatca 4440ttgctgcatg tttggcctcg atgcttccga tggagaaaat aatcaaagga gccttttgcg 4500gtgacgatag tctgctgtac tttccaaagg gttgtgagtt tccggatgtg caacactccg 4560cgaatcttat gtggaatttt gaagcaaaac tgtttaaaaa acagtatgga tacttttgcg 4620gaagatatgt aatacatcac gacagaggat gcattgtgta ttacgatccc ctaaagttga 4680tctcgaaact tggtgctaaa cacatcaagg attgggaaca cttggaggag ttcagaaggt 4740ctctttgtga tgttgctgtt tcgttgaaca attgtgcgta ttacacacag ttggacgacg 4800ctgtatggga ggttcataag accgcccctc caggttcgtt tgtttataaa agtctggtga 4860agtatttgtc tgataaagtt ctttttagaa gtttgtttat agatggctct agttgttaaa 4920ggaaaagtga atatcaatga gtttatcgac ctgacaaaaa tggagaagat cttaccgtcg 4980atgtttaccc ctgtaaagag tgttatgtgt tccaaagttg ataaaataat ggttcatgag 5040aatgagtcat tgtcaggggt gaaccttctt aaaggagtta agcttattga tagtggatac 5100gtctgtttag ccggtttggt cgtcacgggc gagtggaact tgcctgacaa ttgcagagga 5160ggtgtgagcg tgtgtctggt ggacaaaagg atggaaagag ccgacgaggc cattctcgga 5220tcttactaca cagcagctgc aaagaaaaga tttcagttca aggtcgttcc caattatgct 5280ataaccaccc aggacgcgat gagaaacgtc tggcaagttt tagttaatat tagaaatgtg 5340aagatgtcag cgggtttctg tccgctttct ctggagtttg tgtcggtgtg tattgtttat 5400agaaataata taaaattagg tttgagagag aagattacaa acgtgagaga cggagggccc 5460atggaactta cagaagaagt cgttgatgag ttcatggaag atgtccctat gtcgatcagg 5520cttgcaaagt ttcgatctcg aaccggaaaa aagagtgatg tccgcaaagg gaaaaatagt 5580agtagtgatc ggtcagtgcc gaacaagaac tatagaaatg ttaaggattt tggaggaatg 5640agttttaaaa agaataattt aatcgatgat gattcggagg ctactgtcgc cgaatcggat 5700tcgttttaaa tagatcttac agtatcacta ctccatctca gttcgtgttc ttgtcattaa 5760ttaaatggct agcaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt 5820agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgctac 5880atacggaaag cttacactta aatttatttg cactactgga aaactacctg ttccatggcc 5940aacacttgtc actactttct cttatggtgt tcaatgcttt tcccgttatc cggatcatat 6000gaaacggcat gactttttca agagtgccat gcccgaaggt tatgtacagg aacgcactat 6060atctttcaaa gatgacggga actacaagac gcgtgctgaa gtcaagtttg aaggtgatac 6120ccttgttaat cgtatcgagt taaaaggtat tgattttaaa gaagatggaa acattctcgg 6180acacaaactc gagtacaact ataactcaca caatgtatac atcacggcag acaaacaaaa 6240gaatggaatc aaagctaact tcaaaattcg ccacaacatt gaagatggat ccgttcaact 6300agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa 6360ccattacctg tcgacacaat ctgccctttc gaaagatccc aacgaaaagc gtgaccacat 6420ggtccttctt gagtttgtaa ctgctgctgg gattacacat ggcatggatg agctctacaa 6480ataatgacac tcgaggggta gtcaagatgc ataataaata acggattgtg tccgtaatca 6540cacgtggtgc gtacgataac gcatagtgtt tttccctcca cttaaatcga agggttgtgt 6600cttggatcgc gcgggtcaaa tgtatatggt tcatatacat ccgcaggcac gtaataaagc 6660gaggggttcg ggtcgaggtc ggctgtgaaa ctcgaaaagg ttccggaaaa caaaaaagag 6720agtggtaggt aatagtgtta ataataagaa aataaataat agtggtaaga aaggtttgaa 6780agttgaggaa attgaggata atgtaagtga tgacgagtct atcgcgtcat cgagtacgtt 6840ttaatcaata tgccttatac aatcaactct ccgagccaat ttgtttactt aagttccgct 6900tatgcagatc ctgtgcagct gatcaatctg tgtacaaatg cattgggtaa ccagtttcaa 6960acgcaacaag ctaggacaac agtccaacag caatttgcgg atgcctggaa acctgtgcct 7020agtatgacag tgagatttcc tgcatcggat ttctatgtgt atagatataa ttcgacgctt 7080gatccgttga tcacggcgtt attaaatagc ttcgatacta gaaatagaat aatagaggtt 7140gataatcaac ccgcaccgaa tactactgaa atcgttaacg cgactcagag ggtagacgat 7200gcgactgtag ctataagggc ttcaatcaat aatttggcta atgaactggt tcgtggaact 7260ggcatgttca atcaagcaag ctttgagact gctagtggac ttgtctggac cacaactccg 7320gctacttagc tattgttgtg agatttccta aaataaagtc actgaagact taaaattcag 7380ggtggctgat accaaaatca gcagtggttg ttcgtccact taaatataac gattgtcata 7440tctggatcca acagttaaac catgtgatgg tgtatactgt ggtatggcgt aaaacaacgg 7500aaaagtcgct gaagacttaa aattcagggt ggctgatacc aaaatcagca gtggttgttc 7560gtccacttaa aaataacgat tgtcatatct ggatccaaca gttaaaccat gtgatggtgt 7620atactgtggt atggcgtaaa acaacggaga ggttcgaatc ctcccctaac cgcgggtagc 7680ggccca 7686 27 7688 DNA Tobacco mosaic virus 27 gtcgtatttt tacaacaattaccaacaaca acaaacaaca gacaacatta caattactat 60 ttacaattac aatggcatacacacagacag ctaccacatc agctttgctg gacactgtcc 120 gaggaaacaa ctccttggtcaatgatctag caaagcgtcg tctttacgac acagcggttg 180 aagagtttaa cgctcgtgaccgcaggccca aggtgaactt ttcaaaagta ataagcgagg 240 agcagacgct tattgctacccgggcgtatc cagaattcca aattacattt tataacacgc 300 aaaatgccgt gcattcgcttgcaggtggat tgcgatcttt agaactggaa tatctgatga 360 tgcaaattcc ctacggatcattgacttatg acataggcgg gaattttgca tcgcatctgt 420 tcaagggacg agcatatgtacactgctgca tgcccaacct ggacgttcga gacatcatgc 480 ggcacgaagg ccagaaagacagtattgaac tatacctttc taggctagag agagggggga 540 aaacagtccc caacttccaaaaggaagcat ttgacagata cgcagaaatt cctgaagacg 600 ctgtctgtca caatactttccagacatgcg aacatcagcc gatgcagcaa tcaggcagag 660 tgtatgccat tgcgctacacagcatatatg acataccagc cgatgagttc ggggcggcac 720 tcttgaggaa aaatgtccatacgtgctatg ccgctttcca cttctccgag aacctgcttc 780 ttgaagattc atgcgtcaatttggacgaaa tcaacgcgtg tttttcgcgc gatggagaca 840 agttgacctt ttcttttgcatcagagagta ctcttaatta ctgtcatagt tattctaata 900 ttcttaagta tgtgtgcaaaacttacttcc cggcctctaa tagagaggtt tacatgaagg 960 agtttttagt caccagagttaatacctggt tttgtaagtt ttctagaata gatacttttc 1020 ttttgtacaa aggtgtggcccataaaagtg tagatagtga gcagttttat actgcaatgg 1080 aagacgcatg gcattacaaaaagactcttg caatgtgcaa cagcgagaga atcctccttg 1140 aggattcatc atcagtcaattactggtttc ccaaaatgag ggatatggtc atcgtaccat 1200 tattcgacat ttctttggagactagtaaga ggacgcgcaa ggaagtctta gtgtccaagg 1260 atttcgtgtt tacagtgcttaaccacattc gaacatacca ggcgaaagct cttacatacg 1320 caaatgtttt gtccttcgtcgaatcgattc gatcgagggt aatcattaac ggtgtgacag 1380 cgaggtccga atgggatgtggacaaatctt tgttacaatc cttgtccatg acgttttacc 1440 tgcatactaa gcttgccgttctaaaggatg acttactgat tagcaagttt agtctcggtt 1500 cgaaaacggt gtgccagcatgtgtgggatg agatttcgct ggcgtttggg aacgcatttc 1560 cctccgtgaa agagaggctcttgaacagga aacttatcag agtggcaggc gacgcattag 1620 agatcagggt gcctgatctatatgtgacct tccacgacag attagtgact gagtacaagg 1680 cctctgtgga catgcctgcgcttgacatta ggaagaagat ggaagaaacg gaagtgatgt 1740 acaatgcact ttcagaattatcggtgttaa gggagtctga caaattcgat gttgatgttt 1800 tttcccagat gtgccaatctttggaagttg acccaatgac ggcagcgaag gttatagtcg 1860 cggtcatgag caatgagagcggtctgactc tcacatttga acgacctact gaggcgaatg 1920 ttgcgctagc tttacaggatcaagagaagg cttcagaagg tgcattggta gttacctcaa 1980 gagaagttga agaaccgtccatgaagggtt cgatggccag aggagagtta caattagctg 2040 gtcttgctgg agatcatccggaatcgtcct attctaagaa cgaggagata gagtctttag 2100 agcagtttca tatggcgacggcagattcgt taattcgtaa gcagatgagc tcgattgtgt 2160 acacgggtcc gattaaagttcagcaaatga aaaactttat cgatagcctg gtagcatcac 2220 tatctgctgc ggtgtcgaatctcgtcaaga tcctcaaaga tacagctgct attgaccttg 2280 aaacccgtca aaagtttggagtcttggatg ttgcatctag gaagtggtta atcaaaccaa 2340 cggccaagag tcatgcatggggtgttgttg aaacccacgc gaggaagtat catgtggcgc 2400 ttttggaata tgatgagcagggtgtggtga catgcgatga ttggagaaga gtagctgtta 2460 gctctgagtc tgttgtttattccgacatgg cgaaactcag aactctgcgc agactgcttc 2520 gaaacggaga accgcatgtcagtagcgcaa aggttgttct tgtggacgga gttccgggct 2580 gtggaaaaac caaagaaattctttccaggg ttaattttga tgaagatcta attttagtac 2640 ctgggaagca agccgcggaaatgatcagaa gacgtgcgaa ttcctcaggg attattgtgg 2700 ccacgaagga caacgttaaaaccgttgatt ctttcatgat gaattttggg aaaagcacac 2760 gctgtcagtt caagaggttattcattgatg aagggttgat gttgcatact ggttgtgtta 2820 attttcttgt ggcgatgtcattgtgcgaaa ttgcatatgt ttacggagac acacagcaga 2880 ttccatacat caatagagtttcaggattcc cgtaccccgc ccattttgcc aaattggaag 2940 ttgacgaggt ggagacacgcagaactactc tccgttgtcc agccgatgtc acacattatc 3000 tgaacaggag atatgagggctttgtcatga gcacttcttc ggttaaaaag tctgtttcgc 3060 aggagatggt cggcggagccgccgtgatca atccgatctc aaaacccttg catggcaaga 3120 tcttgacttt tacccaatcggataaagaag ctctgctttc aagagggtat tcagatgttc 3180 acactgtgca tgaagtgcaaggcgagacat actctgatgt ttcactagtt aggttaaccc 3240 ctacaccggt ctccatcattgcaggagaca gcccacatgt tttggtcgca ttgtcaaggc 3300 acacctgttc gctcaagtactacactgttg ttatggatcc tttagttagt atcattagag 3360 atctagagaa acttagctcgtacttgttag atatgtataa ggtcgatgca ggaacacaat 3420 agcaattaca gattgactcggtgttcaaag gttccaatct ttttgttgca gcgccaaaga 3480 ctggtgatat ttctgatatgcagttttact atgataagtg tctcccaggc aacagcacca 3540 tgatgaataa ttttgatgctgttaccatga ggttgactga catttcattg aatgtcaaag 3600 attgcatatt ggatatgtctaagtctgttg ctgcgcctaa ggatcaaatc aaaccactaa 3660 tacctatggt acgaacggcggcagaaatgc cacgccagac tggactattg gaaaatttag 3720 tggcgatgat taaaagaaactttaacgcac ccgagttgtc tggcatcatt gatattgaaa 3780 atactgcatc tttggttgtagataagtttt ttgatagtta tttgcttaaa gaaaaaagaa 3840 aaccaaataa aaatgtttctttgttcagta gagagtctct caatagatgg ttagaaaagc 3900 aggaacaggt aacaataggccagctcgcag attttgattt tgtggatttg ccagcagttg 3960 atcagtacag acacatgattaaagcacaac ccaaacaaaa gttggacact tcaatccaaa 4020 cggagtaccc ggctttgcagacgattgtgt accattcaaa aaagatcaat gcaatattcg 4080 gcccgttgtt tagtgagcttactaggcaat tactggacag tgttgattcg agcagatttt 4140 tgtttttcac aagaaagacaccagcgcaga ttgaggattt cttcggagat ctcgacagtc 4200 atgtgccgat ggatgtcttggagctggata tatcaaaata cgacaaatct cagaatgaat 4260 tccactgtgc agtagaatacgagatctggc gaagattggg tttcgaagac ttcttgggag 4320 aagtttggaa acaagggcatagaaagacca ccctcaagga ttataccgca ggtataaaaa 4380 cttgcatctg gtatcaaagaaagagcgggg acgtcacgac gttcattgga aacactgtga 4440 tcattgctgc atgtttggcctcgatgcttc cgatggagaa aataatcaaa ggagcctttt 4500 gcggtgacga tagtctgctgtactttccaa agggttgtga gtttccggat gtgcaacact 4560 ccgcgaatct tatgtggaattttgaagcaa aactgtttaa aaaacagtat ggatactttt 4620 gcggaagata tgtaatacatcacgacagag gatgcattgt gtattacgat cccctaaagt 4680 tgatctcgaa acttggtgctaaacacatca aggattggga acacttggag gagttcagaa 4740 ggtctctttg tgatgttgctgtttcgttga acaattgtgc gtattacaca cagttggacg 4800 acgctgtatg ggaggttcataagaccgccc ctccaggttc gtttgtttat aaaagtctgg 4860 tgaagtattt gtctgataaagttcttttta gaagtttgtt tatagatggc tctagttgtt 4920 aaaggaaaag tgaatatcaatgagtttatc gacctgacaa aaatggagaa gatcttaccg 4980 tcgatgttta cccctgtaaagagtgttatg tgttccaaag ttgataaaat aatggttcat 5040 gagaatgagt cattgtcaggggtgaacctt cttaaaggag ttaagcttat tgatagtgga 5100 tacgtctgtt tagccggtttggtcgtcacg ggcgagtgga acttgcctga caattgcaga 5160 ggaggtgtga gcgtgtgtctggtggacaaa aggatggaaa gagccgacga ggccattctc 5220 ggatcttact acacagcagctgcaaagaaa agatttcagt tcaaggtcgt tcccaattat 5280 gctataacca cccaggacgcgatgaaaaac gtctggcaag ttttagttaa tattagaaat 5340 gtgaagatgt cagcgggtttctgtccgctt tctctggagt ttgtgtcggt gtgtattgtt 5400 tataaaaata atataaaattaggtttgaga gagaagatta caaacgtgag agacggaggg 5460 cccatggaac ttacagaagaagtcgttgat gagttcatgg aagatgtccc tatgtcgatc 5520 aggcttgcaa agtttcgatctcgaaccgga aaaaagagtg atgtccgcaa agggaaaaat 5580 agtagtagtg atcggtcagtgccgaacaag aactatagaa atgttaagga ttttggagga 5640 atgagtttta aaaagaataatttaatcgat gatgattcgg aggctactgt cgccgaatcg 5700 gattcgtttt aaatagatcttacagtatca ctactccatc tcagttcgtg ttcttgtcat 5760 taattaaatg gctagcaaaggagaagaact tttcactgga gttgtcccaa ttcttgttga 5820 attagatggt gatgttaatgggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc 5880 tacatacgga aagcttacccttaaatttat ttgcactact ggaaaactac ctgttccatg 5940 gccaacactt gtcactactttctcttatgg tgttcaatgc ttttcccgtt atccggatca 6000 tatgaaacgg catgactttttcaagagtgc catgcccgaa ggttatgtac aggaacgcac 6060 tatatctttc aaagatgacgggaactacaa gacgcgtgct gaagtcaagt ttgaaggtga 6120 tacccttgtt aatcgtatcgagttaaaagg tattgatttt aaagaagatg gaaacattct 6180 cggacacaaa ctcgagtacaactataactc acacaatgta tacatcacgg cagacaaaca 6240 aaagaatgga atcaaagctaacttcaaaat tcgccacaac attgaagatg gatccgttca 6300 actagcagac cattatcaacaaaatactcc aattggcgat ggccctgtcc ttttaccaga 6360 caaccattac ctgtcgacacaatctgccct ttcgaaagat cccaacgaaa agcgtgacca 6420 catgggcctt cttgagtttgtaactgctgc tgggattaca catggcatgg atgagctcta 6480 caaataatga cactcgaggggtagtcaaga tgcataataa ataacggatt gtgtccgtaa 6540 tcacacgtgg tgcgtacgataacgcatagt gtttttccct ccacttaaat cgaagggttg 6600 tgtcttggat cgcgcgggtcaaatgtatat ggttcatata catccgcagg cacgtaataa 6660 agcgaggggt tcgggtcgaggtcggctgtg aaactcgaaa aggttccgga aaacaaaaaa 6720 gagagtggta ggtaatagtgttaataataa gaaaataaat aatagtggta agaaaggttt 6780 gaaagttgag gaaattgaggataatgtaag tgatgacgag tctatcgcgt catcgagtac 6840 gttttaatca atatgccttatacaatcaac tctccgagcc aatttgttta cttaagttcc 6900 gcttatgcag atcctgtgcagctgatcaat ctgtgtacaa atgcattggg taaccagttt 6960 caaacgcaac aagctaggacaacagtccaa cagcaatttg cggatgcctg gaaacctgtg 7020 cctagtatga cagtgagatttcctgcatcg gatttctatg tgtatagata taattcgacg 7080 cttgatccgt tgatcacggcgttattaaat agcttcgata ctagaaatag aataatagag 7140 gttgataatc aacccgcaccgaatactact gaaatcgtta acgcgactca gagggtagac 7200 gatgcgactg tagctataagggcttcaatc aataatttgg ctaatgaact ggttcgtgga 7260 actggcatgt tcaatcaagcaagctttgag actgctagtg gacttgtctg gaccacaact 7320 ccggctactt agctattgttgtgagatttc ctaaaataaa gtcactgaag acttaaaatt 7380 cagggtggct gataccaaaatcagcagtgg ttgttcgtcc acttaaatat aacgattgtc 7440 atatctggat ccaacagttaaaccatgtga tggtgtatac tgtggtatgg cgtaaaacaa 7500 cggaaaagtc gctgaagacttaaaattcag ggtggctgat accaaaatca gcagtggttg 7560 ttcgtccact taaaaataacgattgtcata tctggatcca acagttaaac catgtgatgg 7620 tgtatactgt ggtatggcgtaaacaacgga gaggttcgaa tcctccccta accgcgggta 7680 gcggccca 7688

What is claimed is:
 1. A capped RNA molecule capable of replication inthe cytoplasm of a host plant cell located in a host plant, comprising:a) a cis-acting viral replication element obtained from asingle-component (+) strand RNA tobamovirus, the plant virus capable ofreplication in the cytoplasm of a host plant cell located in a hostplant; b) a sequence of one to three intervening bases located between acap and the 5′ terminnus of the viral sequence; and c) an exogenous RNAsegment capable of expressing its function in a host plant cell; theexogenous RNA segment being located in a region of said capped RNAmolecule able to tolerate said exogenous RNA segment without disruptingreplication of said capped RNA molecule, wherein said uncapped RNAmolecule is capable of replication in the absence of a trans-actingviral replication element in the host plant cell.
 2. A capped RNAmolecule capable of local spread in a host plant, comprising: a) acis-acting viral replication element obtained from a single-component(+) strand RNA tobamovirus, the plant virus capable of spreading locallyfrom cell to cell in a host plant; b) a sequence of one to threeintervening bases located between a cap and the 5′ terminus of the viralsequence; and c) an exogenous RNA segment capable of expressing itsfunction in a host plant cell; the exogenous RNA segment being locatedin a region of said capped RNA molecule able to tolerate said exogenousRNA segment without disrupting replication of said capped RNA molecule,wherein said uncapped RNA molecule is capable of replication in theabsence of a trans-acting viral replication element in the host plantcell.
 3. A capped RNA molecule capable systemic spread in a host plant,comprising: a) a cis-acting viral replication element obtained from asingle-component (+) strand RNA tobamovirus, the plant virus capablesystemic spread in a host plant; b) a sequence of one to threeintervening bases located between a cap and the 5′ terminus of the viralsequence; and c) an exogenous RNA segment capable of expressing itsfunction in a host plant cell; the exogenous RNA segment being locatedin a region of said capped RNA molecule able to tolerate said exogenousRNA segment without disrupting replication of said capped RNA molecule,wherein said uncapped RNA molecule is capable of replication in theabsence of a trans-acting viral replication element in the host plantcell.